Hetero-Dimeric Immunoglobulins

Abstract

The present invention relates to engineered hetero-dimeric immunoglobulins or fragments thereof and methods of making the same.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 15/597,566, filed May 17, 2017, which is a continuation of U.S. application Ser. No. 14/167,712, filed Jan. 29, 2014, which is a continuation of U.S. application Ser. No. 13/695,773, which is a national stage of Int'l Appl. No. PCT/IB2012/051410, filed Mar. 23, 2012, which claims the benefit of U.S. Provisional Application No. 61/467,727, filed Mar. 25, 2011, which are incorporated by reference herein in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The content of the electronically submitted sequence listing (Name: 3305_0180004_Seqlisting_ST25; Size: 243,284 bytes; and Date of Creation: Feb. 26, 2019) is herein incorporated by reference in its entirety.

THE FIELD OF THE INVENTION

[0003] The present invention relates to engineered hetero-dimeric immunoglobulins or fragments thereof and methods of making the same.

BACKGROUND OF THE INVENTION

[0004] Methods for making hetero-dimeric proteins have been reported. The first approach to construct and produce hetero-dimeric bispecific antibodies was the quadroma technology (Milstein C and Cuello A C, Nature, 305(5934):537-40 (1983)) which consists of a somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different immunoglobulin (Ig) heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different immunogloblin species are generated of which only one is the functional bispecific antibody (Kufer P et al., Trends Biotechnol, 22(5):238-44 (2004)). The presence of mispaired by-products reduces significantly the production yield and requires sophisticated purification procedures to achieve product homogeneity. The mispairing of Ig heavy chains can be reduced by using several rational design strategies, most of which engineer the antibody heavy chains for hetero-dimerization via the design of man-made complementary hetero-dimeric interfaces between the two subunits of the CH3 domain homo-dimer. The first report of an engineered CH3 hetero-dimeric domain pair was made by Carter et al. describing a "protuberance-into-cavity" approach for generating a hetero-dimeric Fc moiety (U.S. Pat. No. 5,807,706; `knobs-into-holes`; Merchant A M et al., Nat Biotechnol, 16(7):677-81 (1998)). Alternative designs have been recently developed and involved either the design of a new CH3 module pair by modifying the core composition of the modules as described in WO2007/110205 or the design of complementary salt bridges between modules as described in WO2007/147901 or WO2009/089004. The disadvantage of the CH3 engineering strategies is that these techniques still result in the production of a significant amount of undesirable homo-dimers. Hence there remains a need for an engineering technique which minimizes the content of homo-dimeric species.

[0005] Regardless of the various approaches at hetero-dimerizing Ig heavy chains as described in these patent publications, the major obstacle facing the development of full bispecific antibodies (i.e., two FAB fragments, each having a unique set of variable heavy and light chain domains that creates a unique antigen binding site and one dimeric Fc region) based on any CH3 domain rational engineering or others, is the requirement of having a common light chain to both FABs in order to circumvent the mispairing of their light chains (Carter P, J Immunol Methods, 248(1-2):7-15 (2001)). Although, this can be accomplished by using antibodies with identical light chains that bind to different antigens by virtue of their distinct heavy chains, it does requires the isolation of such antibodies, which usually involves the use of display technologies, and here are no current technologies that will enable the direct use of two distinct human monoclonal antibodies with the desired specificities to be reassembled without further CDR or light chain engineering into a full bispecific antibody.

[0006] Thus there is a need for generating full, correctly assembled, bispecific antibodies that are similar in their overall structure to natural antibodies, i.e., comprising two FAB fragments, each having a unique set of variable heavy and light chain domains that creates a unique antigen binding site and one dimeric Fc region.

SUMMARY OF THE INVENTION

[0007] The present disclosure relates generally to engineer hetero-dimeric immunoglobulins or fragments thereof and methods of making the same.

[0008] In one aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 12 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 119, the substituted amino acid residue at position 12 and the substituted amino acid residue at position 119 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 26 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 13, the substituted amino acid residue at position 26 and the substituted amino acid residue at position 13 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 27 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 18, the substituted amino acid residue at position 27 and the substituted amino acid residue at position 18 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 79 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 84.3, the substituted amino acid residue at position 79 and the substituted amino acid residue at position 84.3 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at position 11 and the parent domain of the second engineered immunoglobulin chain is a domain which comprises a hinge region, the parent domain of the second engineered immunoglobulin chain is not substituted at position 3 of the hinge region, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0009] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0010] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 86, 88, and 90, wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0011] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residue at position 26 and at a further position selected from the group consisting of 3, 22, 27, 79, 81, 84, 85.1, 86, and 88, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0012] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86 and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3 and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1 and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 86 and 90, wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0013] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86, 88 and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 26 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86, 88 and 90, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0014] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH4 domain, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or wherein the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0015] In another aspect, the present disclosure provides a method to produce a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) substituting at least one amino acid residue of the protein-protein interface of a parent domain of a first parent immunoglobulin chain at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family to obtain a first engineered immunoglobulin chain comprising an engineered domain, (b) substituting at least one amino acid residue of the protein-protein interface of the parent domain of a second parent immunoglobulin chain at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family to obtain a second engineered immunoglobulin chain comprising an engineered domain, (c) culturing a host cell comprising a nucleic acid encoding said engineered immunoglobulin chains, wherein the culturing is such that the nucleic acid is expressed and the engineered immunoglobulin chains produced; and (d) recovering the hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof from the host cell culture.

[0016] In another aspect, the present disclosure provides a method to engineer a protein-protein interface of a domain of a multidomain protein comprising:

(a) providing a multidomain protein comprising a domain with a protein-protein interface; (b) selecting as a donor domain a naturally occurring immunoglobulin super-family member comprising a domain with a protein-protein interface which is different from the domain of (a); (c) overlaying 3D structures of the domain with the protein-protein interface of (a) and the donor domain with the protein-protein interface of (b); (d) identifying exposed protein-protein interface residues in the overlayed 3D structures of the domain with the protein-protein interface of (a) and the donor domain with the protein-protein interface of (b); e) substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) with amino acid residues at the equivalent 3D structural position from the identified exposed protein-protein interface amino acid residues from the donor domain with the protein-protein interface of (b).

[0017] In a further aspect, the present disclosure provides the use of a donor domain of a first and a second member of the naturally occurring immunoglobulin super-family to engineer a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising a first engineered immunoglobulin chain comprising at least one engineered domain which has a protein-protein interface which is substituted with at least one amino acid residue from the protein-protein interface of the donor domain of the first member of the naturally occurring immunoglobulin super-family and a second engineered immunoglobulin chain comprising at least one engineered domain which has a protein-protein interface which is substituted with at least one amino acid residue from the protein-protein interface of the donor domain of the second member of the naturally occurring immunoglobulin super-family.

BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1: Depicts the amino-acid sequence alignment of engineered CH3 domain BT alpha chain and engineered CH3 domain BT beta chain with human IgG1 CH3 domain. EU numbering is used. (Human IgG1CH3 domain: SEQ ID NO: 122; CH3-BT alpha domain: SEQ ID NO: 123; CH3-BT beta domain: SEQ ID NO: 124).

[0019] FIG. 2: Schematic diagram of the various pairings of the BT alpha His and VL-BT beta chains. The BT alpha chain has a polyhistidine tag sequence fused to its C-terminus and BT beta chain has a variable domain antibody fused to its N-terminus.

[0020] FIG. 3: SDS-PAGE analysis demonstrating production of both the BT alpha His_VL-BT beta hetero-dimer and the BT alpha His_VL-BT beta F405S hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) BT alpha His_VL-BT beta transfection output after protein-A purification, non-reducing conditions. (2) BT alpha His_VL-BT beta transfection output after protein-A purification, reducing conditions, (3) Human IgG1-Fc with hinge transfection output after protein-A purification, non-reducing conditions. (4) Human IgG1-Fc with hinge transfection output after protein-A purification, reducing conditions. (5) BT alpha His_VL-BT beta F405S transfection output after protein-A purification, non-reducing conditions. (6) BT alpha His_VL-BT beta F405S transfection output after protein-A purification, reducing conditions. (A) undefined aggregates. (B) BT alpha His_VL-BT beta hetero-dimer. (C) BT alpha His homo-dimer, (D) VL-BT beta F405S homo-dimer. (E) VL-BT beta F405S monomer. (F) BT alpha His monomer. (G) BT alpha His_VL-BT beta F405S hetero-dimer.

[0021] FIG. 4A: Scanning densitometry analysis assessing the relative proportion of the BT alpha His_VL-BT beta hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (A) undefined aggregates. (B) BT alpha His_VL-BT beta hetero-dimer. (C) BT alpha His homo-dimer. FIG. 4B: scanning densitometry analysis assessing the relative proportion of BT alpha His_VL-BT beta hetero-dimer to BT alpha His homo-dimer after protein-A purification when omitting the aggregates (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (A) undefined aggregates. (B) BT alpha His_VL-BT beta hetero-dimer. (C) BT alpha His homo-dimer.

[0022] FIG. 5: SDS-PAGE analysis demonstrating production of the BT alpha His_VL-BT beta F405A hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) BT alpha His_VL-BT beta F405A transaction output after protein-A purification, non-reducing conditions. (2) BT alpha His_VL-BT beta F405A transfection output after protein-A purification, reducing conditions. (3) Human IgG1-Fc with hinge transfection output after protein-A purification, non-reducing conditions. (4) Human IgG1-Fc with hinge transfection output after protein-A purification, reducing conditions. (A) undefined aggregates. (B) VL-BT beta F405A homo-dimer. (C) BT alpha His_VL-BT beta F405A hetero-dimer. (D) BT alpha His homo-dimer. (E) VL-BT beta F405A monomer.

[0023] FIG. 6: Western blot confirming the production of the BT alpha His_VL-BT beta F405A hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). Western blot was detected with an anti His tag antibody. (M) molecular weight marker as indicated. (1) BT alpha His_VL-BT beta F405A transfection output after protein-A purification, non-reducing conditions. (2) BT alpha His_VL-BT beta F405A transfection output after protein-A purification, reducing conditions, (3) BT alpha His_VL-BT beta F405A transfection output after protein-A purification and Ni.sup.2+-sepharose non-reducing conditions. (4) BT alpha His_VL-BT beta F405A transfection output after protein-A purification and Ni.sup.2+-sepharose, reducing conditions. (5) BT alpha His_VL-BT beta F405A transfection output after protein-A purification, Ni.sup.2+-sepharose, and protein-L agarose, non-reducing conditions. (6) BT alpha His_VL-BT beta F405A transfection output after protein-A purification, Ni.sup.2+-sepharose, and protein-L agarose, reducing conditions.

[0024] FIG. 7A: Scanning densitometry analysis assessing the relative proportion of the BT alpha His_VL-BT beta F405A hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (A) undefined aggregates. (B) VL-BT beta F405A homo-dimer. (C) BT alpha His_VL-BT beta F405A hetero-dimer. (D) BT alpha His homo-dimer. (E) VL-BT beta F405A monomer. FIG. 7B: scanning densitometry analysis assessing the relative proportion of the BT alpha His_VL-BT beta F405A hetero-dimer to homo-dimers after protein-A purification when omitting the aggregates (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (A) undefined aggregates. (B) VL-BT beta F405A homo-dimer. (C) BT alpha His_VL-BT beta F405A hetero-dimer. (D) BT alpha His homo-dimer. (E) VL-BT beta F405A monomer.

[0025] FIG. 7C: DSC thermogram of the hetero-dimeric BT alpha His_BT beta (F405A) HA hetero-dimer.

[0026] FIG. 8: SDS-PAGE analysis demonstrating production of the hetero-dimer Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) BT alpha His_VL-BT beta F405A hetero-dimer from example 2, transfection output after protein-A purification, non-reducing conditions. (2) BT alpha His_VL-BT beta F405A hetero-dimer from example 2, transfection output after protein-A purification, reducing conditions. (3) Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S, transfection output after protein-A purification, non-reducing conditions. (4) Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S, transfection output after protein-A purification, reducing conditions. (5) "knob-into-hole" Fc, transfection output after protein-A purification, non-reducing conditions. (6) "knob-into-hole" Fc construct with the "hole" fused to a variable domain antibody, transfection output after protein-A purification, reducing conditions. (A) Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S hetero-dimer. (B) VL-Fc (BTB)-F405A-Y407S homo-dimer. (C) Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N homo-dimer.

[0027] FIG. 9: Scanning densitometry analysis assessing the relative proportion of the hetero-dimer Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (A) Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S. (B) VL-Fc (BTB)-F405A-Y407S homo-dimer. (C) Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N homo-dimer.

[0028] FIG. 10: SDS-PAGE demonstrating production of the BT delta_VL-BT gamma hetero-dimer after ProteinA purification (4-12% SDS Tris-glycine polyacrylamide gel). (M) Molecular weight marker as indicated. (1) BT delta_VL-BT gamma hetero-dimer, transfection output after protein-A purification, reducing conditions. (2) BT delta_VL-BT gamma hetero-dimer, transfection output after protein-A purification, non-reducing conditions. (A) BT delta_VL-BT gamma hetero-dimer. (B) VL-BT gamma homo-dimer. (C) VL-BT gamma monomer.

[0029] FIG. 11A: Scanning densitometry analysis assessing the relative proportion of the BT delta_VL-BT gamma hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). FIG. 11B: Scanning densitometry analysis assessing the relative proportion of the BT delta_VL-BT gamma hetero-dimer after protein-A purification omitting traces of aggregates and monomer in the relative ratio measurements (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions).

[0030] FIG. 12: Schematic diagram of the possible pairings when expressing the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule.

[0031] FIG. 13: SDS-PAGE demonstrating production of the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule (4-12% SDS Tris-glycine polyacrylamide gel). (M) Molecular weight marker as indicated. (1) the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule, transfection output after protein-A purification, non-reducing conditions. (A) the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule. (B) VH(anti-hCD19)-CH1 delta-BT beta c-myc_VL(anti-hCD19)-CK gamma homo-dimer molecule. (C) VH(anti-hCD19)-CH1 delta-BT beta c-myc_VL(anti-hCD19)-CK gamma molecule (half-molecule).

[0032] FIG. 14: Scanning densitometry analysis assessing the relative proportion of the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (1) VH(anti-hCD19)-CH1 delta-BT beta c-myc_VL(anti-hCD19)-CK gamma homo-dimer molecule. (2) the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule. (3) VH(anti-hCD19)-CH1 delta-BT beta c-myc_VL(anti-hCD19)-CK gamma molecule (half-molecule).

[0033] FIG. 15: DSC thermogram of the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule.

[0034] FIG. 16: Schematic diagram of the possible pairings when expressing the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule.

[0035] FIG. 17: SDS-PAGE demonstrating production of the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule (4-12% SDS Tris-glycine polyacrylamide gel). (M) Molecular weight marker as indicated. (1) the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule, transfection output after protein-A purification, non-reducing conditions. (A) the monovalent FAB engineered hetero-dimeric VH(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule. (B) VH(anti-hCD19)-CH13-BT beta c-myc_VL(anti-hCD19)-CK3 homo-dimer molecule. (C) VH(anti-hCD19)-CH13BT beta c-myc_VL(anti-hCD19)-CK3 molecule (half-molecule).

[0036] FIG. 18: scanning densitometry analysis assessing the relative proportion of the monovalent FAB engineered hetero-dimeric VH(anti-hCD9)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (1) VH(anti-hCD19)-CH13-BT beta c-myc_VL(anti-hCD19)-CK3 homo-dimer molecule. (2) the monovalent FAB engineered hetero-dimeric V H(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule. (3) VH(anti-hCD19)-CH13-BT beta c-myc_VL(anti-hCD19)-CK3 molecule (half-molecule).

[0037] FIG. 19A: Sequences of human IGHG1, IGHG2, IGHG3 and IGHG4 CH1 domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution. FIG. 19B: Sequences of human IGHG1 hinge region; the IMGT.RTM. and EU numberings are used; shaded position means no substitution. FIG. 19C: Sequences of human IGHG1, IGHG2, IGHG3 and IGHG4 CH2 domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution; (.about.) indicates a shift in sequence alignment. FIG. 19D: Sequences of human IGHG1, IGHG2, IGHG3 and IGHG4 CH3 domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution.

[0038] FIG. 20A: Sequences of engineered human IGHG1 CH1 domains having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA), TRDC (abbreviated DELTA) TRGC1 (abbreviated GAMMA), and IGHG1 CH3 (abbreviated CH3) constant domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution. FIG. 20B: Sequences of engineered human IGHG1 CH2 domains having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA), TRDC (abbreviated DELTA), TRGC1 (abbreviated GAMMA), and IGIHG1 CH3 (abbreviated CH3) constant domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution. FIG. 20C: Sequences of engineered human IGHG1 CH3 domains having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA), TRDC (abbreviated DELTA), and TRGC1 (abbreviated GAMMA) constant domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution.

[0039] FIG. 21A: Sequences of engineered human IGHE CH4 domains having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA). TRDC (abbreviated DELTA), TRGC1 (abbreviated GAMMA), and IGHG1 CH3 (abbreviated CH3) constant domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution. FIG. 21B: Sequences of engineered human IGHM CH4 domains having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA), TRDC (abbreviated DELTA), TRGC1 (abbreviated GAMMA), and IGHG1 CH3 (abbreviated CH13) constant domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution.

[0040] FIG. 22: Sequences of engineered human IGKC domains having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA). TRDC (abbreviated DELTA), TRGC1 (abbreviated GAMMA), and IGHG1 CH3 (abbreviated CH3) constant domains; the IMGT.RTM. and EU numberings are used; shaded position means no substitution.

[0041] FIG. 23A: Sequences of human IGC1, IGLC2, IGLC3, IGLC6, IGLC7 and engineered variants having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA), TRDC (abbreviated DELTA), TRGC1 (abbreviated GAMMA), and IGHG1 CH3 (abbreviated CH3) constant domains; Kabat residues 108 to 161 are shown--both the IMGT.RTM. and Kabat numberings are used; shaded position means no substitution. FIG. 23B: Sequences of human IGLC1, IGLC2, IGLC3, IGLC6, IGLC7 and engineered variants having protein-protein interfaces based on the human TRAC (abbreviated ALPHA), TRBC2 (abbreviated BETA), TRDC (abbreviated DELTA), TRGC1 (abbreviated GAMMA), and IGHG1 CH3 (abbreviated CH3) constant domains; Kabat residues 162 to 215 are shown--both the IMGT.RTM. and Kabat numberings are used; shaded position means no substitution.

[0042] FIG. 24: Schematic diagram of possible modified heavy chain pairings when expressing the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer.

[0043] FIG. 25: SDS-PAGE demonstrating production of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta. Samples of protein pools after each purification step are shown (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions), (M) Molecular weight marker as indicated. (1) after protein-A chromatography. (2) after anion IEX. (3) after cation IEX. (4) after size-exclusion chromatography. (5) control: scFv-Fc fusion protein. (6) control: monoclonal antibody.

[0044] FIG. 26: Scanning densitometry analysis assessing the relative proportion of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel, non-reducing conditions). (A) VH(anti-EGCFR) CH1-BT alpha_VL(anti-EGFR)-CK homo-dimer. (B) Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer. (C) scFv(anti-HER2)-BT beta homo-dimer. Impurities have been omitted from measurement.

[0045] FIG. 27: DSC thermogram of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer.

[0046] FIG. 28A: BIAcore sensorgram showing the binding and dissociation of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer to/from immobilized human EGFR-Fc.

[0047] FIG. 28B: Demonstration of simultaneous binding of the bispecific Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer to both antigens using SPR technology. The Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer and the human EGFR-Fc were injected sequentially on immobilized human HER2-Fc (solid line). Controls: (dotted line) binding of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer to the immobilized human HER2-Fc, (dashed line) absence of binding of the human EGFR-Fc to the immobilized human HER2-Fc.

[0048] FIG. 29: Schematic diagram of possible modified heavy chain pairings when expressing the anti-hCD19 anti-hVLA2 bispecific antibody. Both heavy chains, VH(anti-hVLA2)-CH1-BT alpha and VH(anti-hCD19)-BT delta-BT beta are C-terminally fused to the His and c-myc tag respectively.

[0049] FIG. 30: SDS-PAGE and Western blot analysis of the anti-hCD19_anti-hVLA2 bispecific antibody. Samples were taken after each purification step (4-12% SDS Tris-glycine polyacrylamide gels under non-reducing conditions); both eluate and unbound pools are shown. (M) Molecular weight marker as indicated. (1) eluate pool from protein-A chromatography. (2) unbound pool from protein-L chromatography. (3) eluate pool from protein-L chromatography. (4) unbound pool from Ni.sup.2+-NTA chromatography. (5) eluate pool from Ni.sup.2+-NTA chromatography. (A) SDS-PAGE. (B) Western blot detected with an anti His tag antibody. (C) Western blot detected with an anti c-myc tag antibody.

[0050] FIG. 31: Flow cytometry detection of the anti-hCD19_anti-hVLA2 bispecific antibody binding to CD19.sup.+ VLA2.sup.- and CD19.sup.- VLA2.sup.+ cells. Stained cells were acquired on a FACS CyAn.TM. ADP flow cytometer. Histograms display the normalized % of maximal event (Y axis) according to Phycoerythrin (PE) fluorescence intensity (X-axis) for each staining.

A and B: Raji cells were stained with the anti-hVLA2 antibody (grey histogram) as an isotype control or the anti-hCD19_anti-VLA2 bispecific antibody (white histogram). Binding of the primary antibodies was revealed by (A) a mouse anti c-myc tag antibody or (B) a mouse anti His tag antibody and stained with a PE-labelled anti-mouse Ig antibody. C and D: HT1080 cells were stained with the anti-hCD19 antibody (grey histogram) as an isotype control or the anti-hCD19_anti-VLA2 bispecific antibody (white histogram). Binding of the primary antibodies was revealed by (C) a mouse anti c-myc tag antibody or (D) a mouse anti His tag antibody and stained with a PE-labelled anti-mouse Ig antibody.

[0051] FIG. 32: DSC thermogram of the anti-hCD19_anti-hVLA2 bispecific antibody.

[0052] FIG. 33: SDS-PAGE analysis demonstrating production of the BT alpha IGHG3_VL-BT beta F405A hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) BT alpha IGHG3_VL-BT beta F405A transfection output after protein-A purification, non-reducing conditions. (2) BT alpha IGHG3_VL-BT beta F405A transfection output after protein-A purification, reducing conditions. (A) VL-BT beta F405A_VL-BT beta F405A homo-dimer. (B) BT alpha IGHG3_VL-BT beta F405A hetero-dimer, (C) VL-BT beta F405A chain (half molecule).

[0053] FIG. 34: Scanning densitometry analysis assessing the relative proportion of the BT alpha IGHG3_VL-BT beta F405A hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel). (A) VL-BT beta F405A_VL-BT beta F405A homo-dimer. (B) BT alpha IGHG3_VL-BT beta F405A hetero-dimer. (C) VL-BT beta F405A chain (half molecule).

[0054] FIG. 35: SDS-PAGE analysis demonstrating production of the Fc IGHAG_VL-Fc IGHGA hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) Fc IGHAG_VL-Fc IGHGA hetero-dimer transfection output after protein-A purification, non-reducing conditions. (2) Fc IGHAG_VL-Fc IGHGA hetero-dimer transfection output after protein-A purification, reducing conditions. (A) VL-Fc IGHGA_VL-Fc IGHGA homo-dimer. (B) Fc IGHAG_VL-Fc IGHGA hetero-dimer. (C) Fc IGHAG_Fc IGHAG homo-dimer. (D) VL-Fc IGHGA chain (half molecule). (E) Fc IGHAG chain (half molecule).

[0055] FIG. 36: Scanning densitometry analysis assessing the relative proportion of the Fc IGHAG_VL-Fc IGHGA hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel). (A) VL-Fc IGHGA_VL-Fc IGHGA homo-dimer. (B) Fc IGHAG_VL-Fc IGHGA hetero-dimer. (C) Fc IGHAG_Fc IGHAG homo-dimer. (D) VL-Fc IGHGA chain (half molecule). (E) Fc IGHAG chain (half molecule).

[0056] FIG. 37: Schematic diagram of engineered CH4 based Fc chains. (A) BT alpha IGHM-4 His chain. (B) VL-BT beta IGHM-4 chain. (C) BT alpha IGHE-4 His chain. (D) VL-BT beta IGHE-4 chain.

[0057] FIG. 38: SDS-PAGE analysis demonstrating production of the BT alpha IGHM-4 His_VL-BT beta IGHM-4 hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) BT alpha IGHM-4 His_VL-BT beta IGHM-4 hetero-dimer transfection output, non-reducing conditions. (2) BT alpha IGHM-4 His_VL-BT beta IGHM-4 hetero-dimer transfection output, reducing conditions. (3) BT alpha IGHM-4 His_VL-BT beta IGHM-4 hetero-dimer transfection output after protein-L purification, non reducing conditions, (4) BT alpha IGHM-4 His_VL-BT beta IGHM-4 hetero-dimer transfection output after protein-L purification, reducing conditions. (A) BT alpha IGHM-4 His_VL-BT beta IGHM-4 beta hetero-dimer.

[0058] FIG. 39: Scanning densitometry analysis assessing the relative proportion of the BT alpha IGHM-4 His_VL-BT IGHM-4 beta hetero-dimer after protein-L purification (4-12% SDS Tris-glycine polyacrylamide gel).

[0059] FIG. 40: SDS-PAGE analysis demonstrating production of the BT alpha IGHE-4 His_VL-BT beta IGHE-4 hetero-dimer (4-12% SDS Tris-glycine polyacrylamide gel). (M) molecular weight marker as indicated. (1) BT alpha IGHE-4 His_VL-BT beta IGHE-4 hetero-dimer transfection output after protein-A purification, non-reducing conditions. (2) BT alpha IGHE-4 His_VL-BT beta IGHE-4 hetero-dimer transfection output after protein-A purification, reducing conditions. (A) BT alpha IGHE-4 His_VL-BT beta IGHE-4 hetero-dimer. (B) BT alpha IGHE-4 His_BT alpha IGHE-4 His homo-dimer. (C) VL-BT beta IGHE-4 chain (half molecule). (D) BT alpha IGHE-4 His chain (half molecule).

[0060] FIG. 41: Scanning densitometry analysis assessing the relative proportion of the BT alpha IGHE-4 His_VL-BT beta IGHE-4 hetero-dimer after protein-A purification (4-12% SDS Tris-glycine polyacrylamide gel).

[0061] FIG. 42: IMGT.RTM. unique numbering as used herein e.g. in example 1, 2, 3, 4, 7, 8, 9, 10, 11, and 12 to identify 3D equivalent positions for human CH1, CH2, CH3, and CH4 constant domains of IGHA1(SEQ ID NO: 98), IGHA2 (SEQ ID NO: 99), IGHD (SEQ ID NO: 100), IGHE (SEQ ID NO: 101), IGHEP1 (SEQ ID NO: 121), IGHG1 (SEQ ID NO: 102), IGHG2 (SEQ ID NO: 103), IGHG3 (SEQ ID NO: 104), IGHG1 (SEQ ID NO: 105), IGHGP (SEQ ID NO: 106), and IGHM (SEQ ID NO: 107). GenBank accession numbers are indicated.

[0062] FIG. 43: IMGT.RTM. unique numbering as used herein e.g. in example 5, 6, 7, and 9 to identify 3D equivalent positions for human IGKC constant domain (SEQ ID NO: 108). GenBank accession number is indicated.

[0063] FIG. 44: IMGT.RTM. unique numbering as used herein e.g. in example 7 to identify 3D equivalent positions for human IGLC1 (SEQ ID NO: 109), IGLC2 (SEQ ID NO: 110), IGLC3 (SEQ ID NO: 111), IGLC6 (SEQ ID NO: 112), and IGLC7 (SEQ ID NO: 113) constant domains. GenBank accession numbers are indicated.

[0064] FIG. 45: IMGT.RTM. unique numbering as used herein e.g. in example 1, 2, 3, 4, 5, 7, 8, 9, 10, and 12 to identify 3D equivalent positions for human TRAC (SEQ ID NO: 114), TRBC1 (SEQ ID NO: 115), TRBC2 (SEQ ID NO: 116), TRDC (SEQ ID NO: 117), TRGC1 (SEQ ID NO: 118), TRGC2 (2.times.) (SEQ ID NO: 119), and TRGC2 (3.times.) (SEQ ID NO: 120) constant domains. GenBank accession numbers are indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The present disclosure relates generally to engineer hetero-dimeric immunoglobulins or fragments thereof and methods of making the same.

[0066] The term "antibody" as referred to herein includes full-length antibodies and any antigen binding fragment or single chains thereof. Antibodies and specifically naturally occurring antibodies are glycoproteins which exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each "Y" shape contains two identical copies of a heavy (H) chain, and two identical copies of a light (L) chain, named as such by their relative molecular weights. Each light chain pairs with a heavy chain, and each heavy chain pairs with another heavy chain. Covalent interchain disulfide bonds and non covalent interactions link the chains together. Antibodies and specifically naturally occurring antibodies contain variable regions, which are the two copies of the antigen binding site. Papain, a proteolytic enzyme splits the "Y" shape into three separate molecules, two so called "Fab" fragments (Fab=fragment antigen binding), and one so called "Fc" fragment or "Fc region" (Fc=: fragment crystallizable). A Fab fragment consists of the entire light chain and part of the heavy chain. The heavy chain contains one variable domain (VH) and either three or four constant domains (CH1, CH2, CH3, and CH4, depending on the antibody class or isotype). The region between the CH1 and CH2 domains is called the hinge region and permits flexibility between the two Fab arms of the Y-shaped antibody molecule, allowing them to open and close to accommodate binding to two antigenic determinants separated by a fixed distance. The "hinge region" as referred to herein is a sequence region of 6-62 amino acids in length, only present in IgA, IgD, and IgG, which encompasses the cysteine residues that bridge the two heavy chains. The heavy chains of IgA, IgD, and IgG each have four domains, i.e. one variable domain (VH) and three constant domains (CH1-3). IgE and IgM have one variable and four constant domains (CH1-4) on the heavy chain. The constant regions of the antibodies may mediate the binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the complement system classical pathway. Each light chain is usually linked to a heavy chain by one covalent disulfide bond.

[0067] Each light chain contains one variable domain (VL) and one light chain constant domain. The light chain constant domain is a kappa light chain constant domain designated herein as IGKC or is a lambda light chain constant domain designated herein as IGLC. IGKC is used herein equivalently to CK or CK and has the same meaning. IGLC is used herein equivalently to Ca or CL and has the same meaning. The term "an IGLC domain" as used herein refer to all lambda light chain constant domains e.g. to all lambda light chain constant domains selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR or FW). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0068] The term "full length antibody" as used herein includes the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, the full length antibody of the IgG class is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and a light chain constant region, and each heavy chain comprising immunoglobulin domains VH, CH1 (C[.gamma.]1), CH2 (C[.gamma.]2), CH3 (C[.gamma.]3), and CH4 (C[.gamma.]4, depending on the antibody class or isotype). In some mammals, for example in camels and llamas, IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to the Fc region.

[0069] Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Human constant light chains are classified as kappa (CK) and lambda (C[.lamda.]) light chains. Heavy chains are classified as mu (.mu.), delta (.delta.), gamma (.gamma.), alpha (.alpha.), or epsilon (.epsilon.), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Thus, "isotype" as used herein is meant any of the classes and/or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3), IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), and IgE (IGHE). The so-called human immunoglobulin pseudo-gamma IGHGP gene represents an additional human immunoglobulin heavy constant region gene which has been sequenced but does not encode a protein due to an altered switch region (Bensmana M et al., Nucleic Acids Res, 16(7):3108 (1988)). In spite of having an altered switch region, the human immunoglobulin pseudo-gamma IGHGP gene has open reading frames for all heavy constant domains (CH1-CH3) and hinge. All open reading frames for its heavy constant domains encode protein domains which align well with all human immunoglobulin constant domains with the predicted structural features. This additional pseudo-gamma isotype is referred herein as IgGP or IGHGP. Other pseudo immunoglobulin genes have been reported such as the human immunoglobulin heavy constant domain epsilon P1 and P2 pseudo-genes (IGHEP1 and IGHEP2). The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgG1, IgG2, IgG3, and IgG4. In mice this class comprises subclasses IgG1, IgG2a, IgG2b, IgG2c and IgG3.

[0070] The term "naturally occurring immunoglobulin super-family" as used herein refers to members of the immunoglobulin super-family which can be found in nature (Williams A F and Barclay A N, Annu Rev Immunol, 6:381-405 (1988)) i.e. which have not been genetically engineered, and includes, but is not limited to Antigen receptors like antibodies; immunoglobulins; T cell receptor chains including the TCR constant domain family; Antigen presenting molecules like Class I MHC, Class II MHC or beta-2 microglobulin; Co-receptors like CD4, CD8, or CD19; Antigen receptor accessory molecules like CD3-.gamma., -.delta. and -.epsilon. chains, CD79a or CD79b; Co-stimulatory or inhibitory molecules like CD28, CD80 or CD86 (also known as B7.1 and B7.2 molecules); Receptors on Natural killer cells like Killer-cell immunoglobulin-like receptors (KIR); Adhesion molecules like CD2, CD48, the SIGLEC family (e.g. CD22, CD83), the CTX family (e.g. CTX, JAMs, BT-IgSF, CAR, VSIG, ESAM), Intercellular adhesion molecules (ICAMs), Vascular cell adhesion molecules (e.g. VCAM-1), Neural Cell Adhesion Molecule (NCAM); Cytokine and growth factor receptors like Interleukin-1 receptor type I, Interleukin-1 receptor type II precursor (IL-1R-2, IL-1R-beta, CD121b antigen), Platelet-derived growth factor receptor (PDGFR), Interleukin-6 receptor alpha chain precursor (IL-6R-alpha, CD126 antigen), Colony stimulating factor 1 receptor precursor (CSF-1-R, CD115 antigen, macrophage colony stimulating factor 1 receptor), Mast/stem cell growth factor receptor precursor (SCFR, c-kit, CD117 antigen), Basic fibroblast growth factor receptor 1 precursor (FGFR-1, Tyrosine kinase receptor CEK1); Receptor tyrosine kinases/phosphatases like Tyrosine-protein kinase receptor Tie-1 precursor or Receptor-type tyrosine-protein phosphatase mu precursor, Ig binding receptors like Polymeric immunoglobulin receptor (PIGR), or selected Fc receptors; and others like CD147, Thymocyte differentiation antigen-1 (Thy-1), also known as CD90, CD7, Butyrophilins (Btn), Sodium channel subunit beta-1 precursor, Titin (a huge intracellular muscle protein also known as Connectin).

[0071] The T cell receptor (TCR) constant domain family as used herein include the human TCR constant domain alpha which is referred herein as "human TCR constant domain alpha" or "TRAC" (SEQ ID NO: 1; GenBank database accession number AAO72258.1 (residues 135-225) which is equivalent to the complete sequence of IMCT.RTM. reference TRAC, the human TCR constant domain beta which is referred herein as "human TCR constant domain beta" or "TRBC2" (SEQ ID NO: 2; GenBank database accession number AAA61026.1 (residues 134-261), which is equivalent to residues 1.8-124 of IMGT.RTM. reference TRBC2), the human TCR constant domain delta which is referred herein as "human TCR constant domain delta" or "TRDC" (SEQ ID NO: 32; GenBank database accession number AAA61125.1 (residues 135-221) which is equivalent to residues 1.7-120 of IMGT.RTM. reference TRDC), the human TCR constant domain gamma which is referred herein as "human TCR constant domain gamma" or "TRGC1" (SEQ ID NO: 33; GenBank database accession number AAA61110.1 (residues 145-245) which is equivalent to residues 1.1-124 of IMGT.RTM. reference TRGC1), and the pre T-cell antigen receptor chains (pre-TCR) as disclosed in Pang S S et al., Nature, 467(7317):844-8 (2010). Within the scope of the invention, allotype variants of the human TCR beta and gamma constant domains (IMGT.RTM. reference TRBC1, and TRGC2 (2.times.) or TRGC2 (3.times.), respectively) are equally included included.

[0072] IMGT.RTM. references are according to IMGT.RTM. (the international ImMunoGeneTics information System.RTM. (Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefings in Functional Genomics &Proteomics, 6(4):253-64 (2007)).

[0073] Preferred naturally occurring immunoglobulin super-family members which can be used for the present invention as donor domains are selected from the group consisting of the human TCR constant domain alpha, the human TCR constant domain beta, the human TCR constant domain gamma, the human TCR constant domain delta, the human TCR variable domain alpha, the human TCR variable domain beta, the human TCR variable domain gamma, the human TCR variable domain delta, pre T-cell antigen receptor chains and the CH1, CH2, CH3, CH4, IGKC, IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 domains. Mom preferred are donor domains selected from the group consisting of the human TCR constant domain alpha, the human TCR constant domain beta, the human TCR constant domain gamma, the human TCR constant domain delta, and the CH3 domain, in particular donor domains selected from the group consisting of the human TCR constant domain alpha, the human TCR constant domain beta, the human TCR constant domain gamma and the human TCR constant domain delta. Preferably naturally occurring immunoglobulin super-family members referred to in the present invention are human naturally occurring immunoglobulins.

[0074] The term "Immunoglobulin fragments" as used herein include, but is not limited to, (i) a domain, (ii) the Fab fragment consisting of VL, VH, CL or CK and CH1 domains, including Fab' and Fab'-SH, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the dAb fragment (Ward E S et al., Nature, 341(6242):544-6 (1989)) which consists of a single variable domain (iv) F(ab').sub.2 fragments, a bivalent fragment comprising two linked Fab fragments (v) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird R E et al., Science, 242(4877):423-6 (1988); Huston J S et al., Proc Natl Acad Sci USA, 85(16):5879-83 (1988)), (vi) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Holliger P et al., Proc Natl Acad Sci USA, 90(14):6444-8 (1993); Tomlinson I and Holliger P, Methods Enzymol, 326:461-79 (2000)), (vii) scFv, diabody or domain antibody fused to an Fc region and (viii) scFv fused to the same or a different antibody.

[0075] The term "hetero-dimeric immunoglobulin" or "hetero-dimeric fragment" or "hetero-dimer" as used herein includes an immunoglobulin molecule or part of comprising at least a first and a second polypeptide, like a first and a second domain, wherein the second polypeptide differs in amino acid sequence from the first polypeptide. Preferably, a hetero-dimeric immunoglobulin comprises two polypeptide chains, wherein the first chain has at least one non identical domain to the second chain, and wherein both chains assemble, i.e. interact through their non identical domains. Specifically, a hetero-dimeric immunoglobulin comprises at least two domains, wherein the first domain is non identical to the second domain, and wherein both domains assemble, i.e. interact through their protein-protein interfaces. More preferably the hetero-dimeric immunoglobulin, has binding specificity for at least two different ligands, antigens or binding sites, i.e. is bispecific. Hetero-dimeric immunoglobulin as used herein includes but is not limited to full length bispecific antibodies, bispecific Fab, bispecific F(ab').sub.2, bispecific scFv fused to an Fc region, diabody fused to an Fc region and domain antibody fused to an Fc region. Preferably, a hetero-dimeric immunoglobulin fragment comprises at least two domains, wherein the first domain is non identical to the second domain, and wherein both domains assemble, i.e. interact through their protein-protein interfaces. More preferably, a hetero-dimeric immunoglobulin fragment comprises at least two engineered domains, wherein the first engineered domain is non identical to the second engineered domain i.e. the first engineered domain differs in amino acid sequence from the second engineered domain, and wherein both engineered domains assemble by interaction through their protein-protein interfaces.

[0076] "Naturally occurring hetero-dimers" as used herein includes but are not limited to an IGKC domain or an IGLC domain (IGLC1 or IGLC2 or IGLC3 or IGLC6 or IGLC7) which hetero-dimerizes with any heavy chain CH1 domain (IGHA1 CH1, IGHA2 CH1, IGHD CH1, IGHE CH1, IGHG1 CH1, IGHG2 CH1, IGHG3 CH1, IGHG4 CH1, IGHGP CH1, IGHM CH1), e.g. IGKC/IGHA1 CH1, IGLC2/IGHA1 CH1, IGLC3/IGHA1 CH1, IGLC6/IGHA1 CH1, IGLC7/IGHA1 CH1, IGKC/IGHA2 CH1, IGLC2/IGHA2 CH1, IGLC3/IGHA2 CH1, IGLC6/IGHA2 CH1, IGLC7/IGHA2 CH1, IGKC/IGHD CH1, IGLC2/IGHD CH1, IGLC3/IGHD CH1, IGLC6/IGHD CH1, IGLC7/IGHD CH1, IGKC/IGHE CH1, IGLC2/IGHE CH1, IGLC3/IGHE CH1, IGLC6/IGHE CH1, IGLC7/IGHE CH1, IGKC/IGHG1 CH1, IGLC2/IGHG1 CH1, IGLC3/IGHG1 CH1, IGLC6/IGHG1 CH1, IGLC7/IGHG1 CH1, IGKC/IGHG2 CH1, IGLC2/IGHG2 CH1, IGLC3/IGHG2 CH1, IGLC6/IGHG2 CH1, IGLC7/IGHG2 CH1, IGKC/IGHG3 CH1, IGLC2/IGHG3 CH1, IGLC3/IGHG3 CH1, IGLC6/IGHG3 CH1, IGLC7/IGHG3 CH1, IGKC/IGHG4 CH1, IGLC2/IGHG4 CH1, IGLC3/IGHG4 CH1, IGLC6/IGHG4 CH1, IGLC7/IGHG4 CH1, IGKC/IGHGP CH1, IGLC2/IGHGP CH1, IGLC3/IGHGP CH1, IGLC6/IGHGP CH1, IGLC7/IGHGP CH1, IGKC/IGHM CH1, IGLC2/IGHM CH1, IGLC3/IGHM CH1, IGLC6/IGHM CH1, IGLC7/IGHM CH1. Other examples of "naturally occurring hetero-dimers" encompass an antibody variable heavy chain domain which hetero-dimerizes with an antibody variable light chain domain (kappa or lambda), a TCR alpha variable domain which hetero-dimerizes with a TCR beta variable domain, a TCR gamma variable domain which hetero-dimerizes with a TCR delta variable domain, a TCR alpha constant domain which hetero-dimerizes with a TCR beta constant domain, a TCR gamma constant domain which hetero-dimerizes with a TCR delta constant domain.

[0077] The term "homo-dimeric immunoglobulin" or "homo-dimeric fragment" or "homo-dimer" as used herein includes an immunoglobulin molecule or part of comprising at least a first and a second polypeptide, like a first and a second domain, wherein the second polypeptide is identical in amino acid sequence to the first polypeptide. Preferably, a homo-dimeric immunoglobulin comprises two polypeptide chains, wherein the first chain has at least one identical domain to the second chain, and wherein both chains assemble, i.e. interact through their identical domains. Specifically, a homo-dimeric immunoglobulin comprises at least two identical domains and wherein both domains assemble, i.e. interact through their protein-protein interfaces. Preferably, a homo-dimeric immunoglobulin fragment comprises at least two domains, wherein the first domain is identical to the second domain, and wherein both domains assemble, i.e. interact through their protein-protein interfaces. "Naturally occurring homo-dimers" as used herein include but are not limited to e.g. two CH3 domains of the same species, isotype and subclass e.g. human IGHG1 CH3/IGHG1 CH3, human IGHG2 CH3/IGHG2 CH3, human IGHG3 CH3/IGHG3 CH3, human IGHG4 CH3/IGHG4 CH3, human IGHA1 CH3/IGHA1 CH3, human IGHA2 CH3/IGHA2 CH3, human IGHE CH3/IGHE CH3, human IGHEP1 CH3/IGHEP1 CH3, human IGHM CH3/IGHM CH3, human IGHD CH3/IGHD CH3, human IGHGP CH3/IGHGP CH3, two CH2 domains of the same species, isotype and subclass e.g. human IGHG1 CH2/IGHG1 CH2, human IGHG2 CH2/IGHG1 CH2, human IGHG3 CH2/IGHG3 CH2, human IGHG4 CH2/IGHG4 CH2, human IGHA1 CH2/IGHA1 CH2, human IGHA2 CH2/IGHA2 CH2, human IGHE CH2/IGHE CH2, human IGHEP1 CH2/IGHEP1 CH2, human IGHM CH2/IGHM CH2, human IGHD CH2/IGHD CH2, human IGHGP CH2/IGHGP CH2, or two CH4 domains of the same species, isotype and subclass e.g. human IGHE CH4/GHE CH4, human IGHM CH4/IGHM CH4. Preferred "Naturally occurring homo-dimers" are selected from the group consisting of human IGHG1 CH3/IGHG1 CH3, human IGHG2 CH3/IGHG2 CH3, human IGHG3 CH3/IGHG3 CH3, human IGHG4 CH3/IGHG4 CH3, human IGHA1 CH3/IGHA1 CH3, human IGHA2 CH3/IGHA2 CH3, human IGHE CH3/IGHE CH3, human IGHM CH3/IGHM CH3, human IGHD CH3/IGHD CH3, human IGHGP CH3/IGHGP CH3, human IGHE CH2/IGHE CH2, human IGHM CH2/IGHM CH2, human IGHE CH4/IGHE CH4, and human IGHM CH4/IGHM CH4.

[0078] Most immunoglobulin light chains associate into dimers (Novotny J and Haber E, Proc Natl Acad Sci USA, 82(14):4592-6 (1985)). Both kappa and lambda light chains have been reported to homo-dimerize, and several crystal structures of kappa and lambda light chain dimers are available from the Protein Data Bank (PDB) database (Roussel A et al., Eur J Biochem, 260(1):192-9 (1999), Huang D B et al., Proc Natl Acad Sci USA, 93(14):7017-21 (1996); www.pdb.org; Bernstein F C et al., Eur J Biochem, 80(2):319-24 (197T)). Within the scope of the invention homo-dimers of an IGKC domain or an IGLC domain (IGLC1 or IGLC2 or IGLC3 or IGLC6 or IGLC7) can be considered as parent or donor domains for protein-protein interface engineering. Thus, "naturally occurring homo-dimers" as used herein also include but are not limited to two IGKC domains of the same species, isotype and subclass e.g. human IGKC/IGKC, two IGLC domains of the same species, isotype and subclass e.g. human IGLC1/IGLC1, human IGLC2/IGLC2, human IGLC3/IGLC3, human IGLC6/IGLC6, human IGLC7/IGLC7 and any other pairwise combination of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7.

[0079] The term "formation of a hetero-dimer" or "forming a hetero-dimer" or "capable to form a hetero-dimer" in relation to two domains e.g. in relation to two parent domains or to two donor domains as used herein means that the first domain is not identical to the second domain and wherein both domains assemble, i.e. interact through their protein-protein interface wherein the protein-protein interfaces are normally not identical. The first domain is not identical to the second domain if both domains differ in amino acid sequence by at least one amino acid residue. The protein-protein interfaces are normally not identical if the protein-protein interface of the first parent or donor domain has at least one 3D structural position with a different amino acid residue compared to the protein-protein interface of the second parent or donor domain.

[0080] The term "formation of a naturally occurring hetero-dimer" or "forming a naturally occurring hetero-dimer" or "capable to form a naturally occurring hetero-dimer" in relation to two domains e.g. in relation to two naturally occurring parent domains or to two naturally occurring donor domains as used herein means that the first domain is not identical to the second domain and wherein both domains assemble, i.e. interact through their protein-protein interface wherein the protein-protein interfaces are normally not identical. The first domain is not identical to the second domain if both domains differ in amino acid sequence by at least one amino acid residue. The protein-protein interfaces are normally not identical if the protein-protein interface of the first parent or donor domain has at least one 3D structural position with a different amino acid residue compared to the protein-protein interface of the second parent or donor domain.

[0081] The term "formation of a homo-dimer" or "forming a homo-dimer" or "capable to form a homo-dimer" in relation to two domains e.g. in relation to two parent domains or to two donor domains as used herein means that the first domain is identical to the second domain and wherein both domains assemble, i.e. interact through their protein-protein interface, wherein the protein-protein interfaces are normally identical. The first domain is identical to the second domain if their amino acid sequences are identical. The protein-protein interfaces are normally identical if the protein-protein interface of the first parent or donor domain has the identical amino acid at the identical 3D structural position compared to the protein-protein interface of the second parent or donor domain.

[0082] The term "formation of a naturally occurring homo-dimer" or "forming a naturally occurring homo-dimer" or "capable to form a naturally occurring homo-dimer" in relation to two domains e.g. in relation to two naturally occurring parent domains or to two naturally occurring donor domains as used herein means that the first domain is identical to the second domain and wherein both domains assemble, i.e. interact through their protein-protein interface, wherein the protein-protein interfaces are normally identical. The first domain is identical to the second domain if their amino acid sequences are identical. The protein-protein interfaces are normally identical if the protein-protein interface of the first parent or donor domain has the identical amino acid at the identical 3D structural position compared to the protein-protein interface of the second parent or donor domain.

[0083] The term "domain" as used herein includes any region of a polypeptide that is responsible for selectively assembling with a protein partner (i.e., another protein (or region of) or another domain) and/or can perform a complete biological function or part of like binding a receptor, or a substrate, independently or within a multidomain entity. Usually a domain as referred to herein is not a hinge region and/or does not contain a hinge region. The domain can exist independently of the rest of a protein chain. A domain forms a compact three-dimensional structure and is independently stable and folded. Domains vary in length from between about amino acids up to 500 amino acids in length. Preferably the domains as used herein vary in length from between about 70 amino acids up to about 120 amino acids in length. The shortest domains such as zinc fingers are stabilized by metal ions or disulfide, bridges. Domains often form functional units, such as the calcium-binding EF hand domain of calmodulin. Because they are self-stable, domains can be "swapped" by genetic engineering between one protein and another to make chimeric proteins. Immunoglobulins are made of variable and constant domains belonging to the immunoglobulin superfamily (Williams A F and Barclay A N, Annu Rev Immunol, 6:381-405 (1988); Bork P et al., J Mol Biol, 242(4); 309-20 (1994)). Domains, which are included herein are CH1 and CH3, specifically naturally occurring CH1 and CH3, from IGHA1, IGHA2, IGHD, IGHE, IGHG1, IGHG2, IGHG3, IGH4, IGHGP and IGHM; CH4, specifically naturally occurring CH4, from IGHE and IGHM; IGKC, IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7, specifically naturally occurring IGKC, IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7. Further domains which are included herein are CH2, specifically naturally occurring CH2, from IGHA1, IGHA2, IGHD, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP and IGHM. Preferably unglycosylated CH2 domains are used herein which in their unglycosylated form are homo-dimers as referred herein. The CH2 domains in an unglycosylated Fc fragment approach each other much more closely compared to the CH2 domains in a naturally glycosylated Fc fragment. The crystal structure of the murine unglycosylated IgG1 Fc fragment has shown that a fully unglycosylated Fc fragment can adopt a "closed" structure with the distance between the Pro 332 from the CH2 domain of the first unglycosylated immunoglobulin chain and the Pro 332 from the CH2 domain of the second unglycosylated immunoglobulin chain is only 11.6 .ANG. (Feige M J et al., J Mol Biol, 391(3):599-608 (2009)). Further domains which are included herein are VH and VL domains which do not have an engineered protein-protein interface according to the invention, i.e. which are not specifically engineered to modify its naturally occurring protein-protein interface except for backmutations arising out of the humanization process. The term "naturally occurring domain" as used herein refers to domains which can be found in nature i.e. which have not been genetically engineered.

[0084] For all immunoglobulin constant domains included in the present invention, numbering can be according to the IMGT.RTM. (IMGT.RTM., the international ImMunoGencTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0085] For all human CH1, CH2, CH3 immunoglobulin heavy chain constant domains selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, numbering can be according to the "EU numbering system" (Edelman G M et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). The correspondence between the IMGT.RTM. unique numbering and the EU numbering for all human CH1, CH2, CH3 immunoglobulin heavy chain constant domains selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4 is shown in FIG. 19. A complete correspondence for the human CH1, hinge, CH2, and CH3 constant regions of IGHG1 can be found at the IMGT.RTM. database (IMGT.RTM., the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Rev, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005)); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0086] For the human kappa immunoglobulin light chain constant domain (IGKC), numbering can be according to the "EU numbering system" (Edelman G M et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). The correspondence between the IMGT.RTM. unique numbering and the EU numbering for the human IGKC immunoglobulin light chain constant domain is shown in FIG. 22. A complete correspondence for the human CK domain can be found at IMGT database (IMGT.RTM., the international ImMunoGeneTics information System.RTM.; Lefranc M P et al, Nucleic Acids Rev, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1); 219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005)); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0087] For the human lambda immunoglobulin light chain constant domains (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7), numbering can be according to the "Kabat numbering system" (Kabat E A et al., Sequences of proteins of immunological interest. 5.sup.th Edition--US Department of Health and Human Services, NIH publication no. 91-3242 (1991)) as described by Dariavach P et al., Proc Natl Acad Sci USA, 84(24):9074-8 (1987) and Frangione B et al., Proc Natl Acad Sci USA, 82(10):3415-9 (1985). The correspondence between the IMGT unique numbering and the Kabat numbering for human immunoglobulin light chain constant domains (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7) is shown in FIG. 23. A complete correspondence for human IGLC domains can be found at the IMGT database (IMGT.RTM., the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3); 185-203 (2005)); Kaas Q et al., Briefing in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0088] The human IGHG1 immunoglobulin heavy chain constant domains referred herein as the following domain boundaries: CH1 [EU numbering: 118-215], Hinge .gamma.1 [EU numbering: 216-2301, CH2 [EU numbering: 231-340], and CH3 (EU numbering: 341-447]. The human CK domain referred herein spans residues 108 to 214 (EU numbering). The human IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 domains referred herein span residues 108-215 (Kabat numbering).

[0089] A "multidomain entity" or "multidomain protein" as used herein comprises at least two domains. These two domains can be on one immunoglobulin chain (one polypeptide) or can be on two immunoglobulin chains (two polypeptides).

[0090] The term "engineered immunoglobulin chain" as used herein includes an immunoglobulin chain comprising at least one engineered domain with a protein-protein interface which differs from the parent domain.

[0091] The term "parent immunoglobulin chain" as used herein includes any existing immunoglobulin chain, specifically naturally occurring immunoglobulin chains, which can be used as a parent sequence for designing an engineered immunoglobulin chain. Preferably, the the parent immunoglobulin chains, e.g. the parent immunoglobulin chain of the first engineered immunoglobulin chain and/or the parent immunoglobulin chain of the second engineered immunoglobulin chain and/or the parent immunoglobulin chain of the third engineered immunoglobulin chain are naturally occurring immunoglobulin chains.

[0092] The term "parent domain" as used herein includes any existing domain, specifically naturally occurring domains, more specifically naturally occurring domains which form a naturally occurring hetero-dimer or a naturally occurring homo-dimer, which can be used as a parent sequence for designing an engineered domain. The parent domain of the engineered domain of the first, second, third and fourth engineered immunoglobulin chain is not a zinc finger. The parent domain is usually an immunoglobulin domain, e.g. a domain of an immunoglobulin chain. Preferably, the parent domains, e.g. the parent domain of a first and/or a second engineered immunoglobulin chain and/or the parent domain of a further engineered domain of the first and/or the second engineered immunoglobulin chain and/or the parent domain of an engineered domain of a third engineered immunoglobulin chain are naturally occurring domains.

[0093] The term "donor domain" as used herein includes naturally occurring domains, specifically naturally occurring domains which form a naturally occurring hetero-dimer or a naturally occurring homo-dimer, which can be used as a donor sequence for designing an engineered domain, i.e. which can be used as donor sequence for substituting amino acid residues at the equivalent 3D structural position in a parent domain.

[0094] The term "engineered domain" as used herein includes a domain engineered from a parent domain and a donor domain.

[0095] The term "protein-protein interface" as used herein includes amino acid residues that mediate direct-contact association of a protein domain with amino acid residues of another protein domain thereby defining a 3D interface. These amino acid residues that mediate direct-contact association between two domains include any amino acid from one partner interacting with one or more amino acid from the other partner. The term "interacting" in relation to interacting amino acid residues as used herein includes any amino acid from one partner having at least one heavy atom that is less than 15 .ANG. away from any heavy atom of an amino acid residue in the other partner. The term "heavy atom of an amino acid residue" refers herein to any atom of an amino acid residue which is not a hydrogen atom. Preferably, interacting amino acid residues include any amino acids from one partner having at least one heavy atom that is less than 10 .ANG. away from any heavy atom of an amino acid residue in the other partner. Most preferably, interacting amino acid residues include any amino acids from one partner having at least one heavy atom that is less than 5 .ANG. away from any heavy atom of an amino acid residue in the other partner. The interaction of amino acid residues can be mediated by forces which include van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or electrostatic forces, hydrophobic contacts, and disulfide bonds or other forces known to one skilled in the art. Protein-protein interfaces are more hydrophobic and bury twice as much protein surfaces as in protein complexes, and usually assemble at the time they fold (Bahadur R P et al, Proteins, 53(3):708-19 (2003)). Analysis of 47 FAB fragments experimentally determined 3D structures showed that up to 50 residues in the constant domain light chain and up to 52 residues in the constant domain heavy chain compose the domain protein-protein interfaces (Potapov V et al., J Mol Biol, 342(2):665-79 (2004)). The CH3 protein interface involves 16 residues located on four antiparallel beta-strands that make intermolecular contacts and are buried 1090 .ANG.2 from each surface (Dall'Acqua W et al., Biochemistry, 37(26):9266-73 (1998)).

[0096] The protein-protein interfaces of the engineered domains are considered to interact by homo-dimerization if the protein-protein interface of the donor domain of e.g. the first member of the naturally occurring immunoglobulin super-family and the protein-protein interface of the donor domain of e.g. the second member of the naturally occurring immunoglobulin super-family interact by homo-dimerization, e.g. if both donor domains form a homo-dimer, and if all amino acid residues of the protein-protein interfaces of the parent domains are substituted with amino acid residues of the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family. Thus protein-protein interfaces of the engineered domains are considered to interact by homo-dimerization if all amino acid residues of the protein-protein interface of the first engineered domain are identical to the amino acid residues of the protein-protein interface of the second engineered domain at identical 3D positions. The protein-protein interfaces of the engineered domains are considered to interact by hetero-dimerization if the protein-protein interface of the donor domain of e.g. the first member of the naturally occurring immunoglobulin super-family and the protein-protein interface of the donor domain of e.g. the second member of the naturally occurring immunoglobulin super-family interact by homo-dimerization, e.g. if both donor domains form a homo-dimer, and if not all amino acid residues of the protein-protein interfaces of the parent domains are substituted with amino acid residues of the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family. More specifically the protein-protein interfaces of the engineered domains are considered to interact by hetero-dimerization if the protein-protein interface of the donor domain of e.g. the first member of the naturally occurring immunoglobulin super-family and the protein-protein interface of the donor domain of e.g. the second member of the naturally occurring immunoglobulin super-family interact by homo-dimerization, e.g. if both donor domains form a homo-dimer, and if at least one amino acid residue in the first parent domain is substituted with an amino acid residue at the 3D equivalent structural position in the donor domain of the first member of the naturally occurring immunoglobulin super-family and wherein the second parent domain is not substituted with the identical amino acid residue which was substituted in the first parent domain at the identical 3D structural position.

[0097] The protein-protein interfaces of the engineered domains are considered to interact by hetero-dimerization if the protein-protein interface of the donor domain of e.g. the first member of the naturally occurring immunoglobulin super-family and the protein-protein interface of the donor domain of e.g. the second member of the naturally occurring immunoglobulin super-family interact by hetero-dimerization e.g. if both donor domains form a hetero-dimer, and if all or not all amino acid residues of the protein-protein interfaces of the parent domains are substituted with amino acid residues of the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family.

[0098] Thus protein-protein interfaces of the engineered domains are considered to interact by hetero-dimerization if not all amino acid residues of the protein-protein interface of the first engineered domain are identical to the amino acid residues of the protein-protein interface of the second engineered domain at identical 3D positions.

[0099] The term "equivalent 3D structural position" or "equivalent 3D position" or "3D equivalent structural position" or "3D equivalent position" are used interchangeably herein and include the position of an amino acid residue in the protein-protein interface of a donor domain which when overlaid on the protein-protein interface of a parent domain by superimposing the carbon alpha traces of both domains, occupies a 3D position within less than a distance of 6 .ANG. to the closest residue of the parent domain, wherein the donor domain is different from the parent domain. Superimposition can be performed by comparing the coordinates of the atoms in the same frame of reference. Beside the carbon alpha traces, backbone atoms (N, C and O) of both domains can be superimposed. The 3D structural positions can be selected as well based on visual inspection (i.e. intuition based design or knowledge based design) or positions can be selected after enumerating energy terms like van der Waals interaction, hydrogen bond interaction etc. (i.e. rational design). 3D structures of the domains as used herein includes experimentally solved 3D structure in crystalline or solution state which can be retrieved the Protein Data Bank (www.pdb.org; Bernstein F C et al., Eur J Biochem, 80(2):319-24 (1977); Berman H M et al., Nucleic Acids Res, 28(1):235-42 (2000)) or other databases. In addition, 3D structures of the domains which are not available from public databases or have not been solved can be modelled using a number of computational methods such as Ab initio prediction methods where only the amino acid sequence of the protein is required or threading and homology modeling methods where a 3D model for the protein domain is build from experimental structures of evolutionary related proteins (Zhang Y, Curr Opin Struct Biol, 18(3):342-8 (2008)). Without being bound by theory, alternatively the IMGT.RTM. numbering system can be used to identify equivalent 3D structural positions in immunoglobulin domains since this system is based on the comparative analysis of the 3D structure of the immunoglobulin super-family domains.

[0100] The term "Non essential to the core integrity of the domain" as used herein includes residues which are non-essential for correct folding and/or the hydrophobic core of the protein domain. Folded proteins usually have a hydrophobic core in which side chain packing stabilizes the folded state, and charged or polar side chains occupy the solvent-exposed surface where they interact with surrounding water, ions or others ligands such as other proteins or protein domains or carbohydrates. Formation of intramolecular hydrogen bonds provides another important contribution to protein core stability. Non essential residues to the core integrity of a protein domain include but are not limited to non-proline residues, non cysteine residues involved in an intramolecular disulfide bond, exposed residues, and residues which are not involved intramolecular hydrogen bonds.

[0101] The terms "protein" and "polypeptide" as used herein have the same meaning and are used interchangeably.

[0102] The terms "amino acid" or "amino acid residue" as used herein includes natural amino acids as well as non-natural amino acids. Preferably natural amino acids are included.

[0103] The term "charged amino acid" as used herein includes the amino acids lysine (positively charged), arginine (positively charged), histidine (positively charged), aspartic acid (negatively charged) and glutamic acid (negatively charged). Normally, charged amino acid as used herein are lysine, arginine, histidine, aspartic acid and glutamic acid. The term "charged pair" or "charged pair of amino acids" or "charged amino acid pair" have the same meaning and are used interchangeably and include any combination of one positively charged amino acid with one negatively charged amino acid. This includes the following pairs of charged amino acids: lysine/aspartic acid, lysine/glutamic acid, arginine/aspartic acid, arginine/glutamic acid, histidine/aspartic acid, and histidine/glutamic acid.

[0104] The terms "substitution" or "amino acid substitution" or "amino acid residue substitution" as used herein refers to a substitution of a first amino acid residue in an amino acid sequence with a second amino acid residue, whereas the first amino acid residue is different from the second amino acid residue i.e. the substituted amino acid residue is different from the amino acid which has been substituted.

[0105] The term "amino acid residues which are not adjacent" or "not adjacent amino acid residues" as used herein refers to two amino acids within an amino acid sequence which are not immediately adjoining i.e. which have at least one intervening amino acid residue in between them.

[0106] The term "Fab" or "Fab region" as used herein includes the polypeptides that comprise the VH, CH1, VL, and light chain constant immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment.

[0107] The term "Fc" or "Fc region", as used herein includes the polypeptide comprising the constant region of an antibody heavy chain excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C[.gamma.]2 and C[.gamma.]3) and the hinge between Cgamma1 (C[.gamma.]1) and Cgamma2 (C[.gamma.]2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl terminus, wherein the numbering is according to the EU index. Fc may refer to this region in isolation or this region in the context of an Fc polypeptide, for example an antibody.

[0108] The term "amino acid modification" herein includes an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution R94K refers to a variant polypeptide, in which the arginine at position 94 is replaced with a lysine. For example 94K indicates the substitution of position 94 with a lysine. For the purposes herein, multiple substitutions are typically separated by a slash or a comma. For example, "R94K/L78V" or "R94K, L78V" refers to a double variant comprising the substitutions R94K and L78V. By "amino acid insertion" or "insertion" as used herein is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. For example, insert -94 designates an insertion at position 94. By "amino acid deletion" or "deletion" as used herein is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. For example, R94-- designates the deletion of arginine at position 94.

[0109] The term "conservative modifications" or "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0110] "Conservative amino acid substitutions" as used herein includes amino acid substitutions in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Hetero-Dimeric Immunoglobulins

[0111] In one aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 12 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 119, the substituted amino acid residue at position 12 and the substituted amino acid residue at position 119 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 26 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 13, the substituted amino acid residue at position 26 and the substituted amino acid residue at position 13 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 27 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 18, the substituted amino acid residue at position 27 and the substituted amino acid residue at position 18 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 79 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 84.3, the substituted amino acid residue at position 79 and the substituted amino acid residue at position 84.3 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGKC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at position 11 and the parent domain of the second engineered immunoglobulin chain is a domain which comprises a hinge region, the parent domain of the second engineered immunoglobulin chain is not substituted at position 3 of the hinge region, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0112] Preferably, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 12 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 119, the substituted amino acid residue at position 12 and the substituted amino acid residue at position 119 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 26 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 13, the substituted amino acid residue at position 26 and the substituted amino acid residue at position 13 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 27 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 18, the substituted amino acid residue at position 27 and the substituted amino acid residue at position 18 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the unsubstituted or substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 79 and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 84.3, the unsubstituted or substituted amino acid residue at position 79 and the substituted amino acid residue at position 84.3 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at position 11 and the parent domain of the second engineered immunoglobulin chain is a domain which comprises a hinge region, the parent domain of the second engineered immunoglobulin chain is not substituted at position 3 of the hinge region, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0113] Even more preferably, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 12 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 119, the substituted amino acid residue at position 12 and the substituted amino acid residue at position 119 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 26 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 13, the substituted amino acid residue at position 26 and the substituted amino acid residue at position 13 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 27 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 18, the substituted amino acid residue at position 27 and the substituted amino acid residue at position 18 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 79 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 84.3, the substituted amino acid residue at position 79 and the substituted amino acid residue at position 84.3 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at position 11 and the parent domain of the second engineered immunoglobulin chain is a domain which comprises a hinge region, the parent domain of the second engineered immunoglobulin chain is not substituted at position 3 of the hinge region, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0114] Position 5 of an IGLC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 116 according to Kabat numbering.

[0115] Position 12 of an IGLC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 123 according to Kabat numbering.

[0116] Position 13 of an IGLC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 124 according to Kabat numbering.

[0117] Position 18 of an IGLC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 129 according to Kabat numbering.

[0118] Position 20 of an IGLC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 131 according to Kabat numbering.

[0119] Position 79 of an IGLC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 160 according to Kabat numbering.

[0120] Position 11 of the IGKC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 122 according to EU numbering.

[0121] Position 12 of the IGKC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 123 according to EU numbering.

[0122] Position 20 of an IGKC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 131 according to EU numbering.

[0123] Position 86 of an IGKC domain in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 176 according to EU numbering.

[0124] Position 20 of a CH1 domain in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 141 according to EU numbering.

[0125] Position 26 of a CH1 domain in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 147 according to EU numbering.

[0126] Position 27 of a CH1 domain in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 148 according to EU numbering.

[0127] Position 84.3 of a CH1 domain in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 176 according to EU numbering.

[0128] Position 86 of the CH1 domain in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 183 according to EU numbering.

[0129] Position 119 of a CH1 domain in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain indicated according to the IMGT.RTM. numbering correspond to position 213 according to EU numbering.

[0130] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0131] Preferably the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the unsubstituted or substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0132] Even more preferably the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not charged amino acids, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0133] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0134] Preferably the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the unsubstituted or substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0135] Even more preferably the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH) domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not charged amino acids, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0136] In some aspects the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 5, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, preferably are not charged amino acids, and wherein, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, preferably are not charged amino acids, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0137] In some aspects the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and wherein, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, preferably are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, preferably are not charged amino acids, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 5, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, preferably are not charged amino acids, and wherein, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, preferably are not charged amino acids, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0138] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 86, 88, and 90, wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0139] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7.22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residue at position 26 and at a further position selected from the group consisting of 3, 22, 27, 79, 81, 84, 85.1, 86, and 88, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0140] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86 and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3 and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1 and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 86 and 90, wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0141] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86, 88 and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 26 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86, 88 and 90, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0142] In a further aspect, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH4 domain, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or wherein the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0143] In some embodiments the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer. In some embodiments the donor domain of the third member of the naturally occurring immunoglobulin super-family and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer.

[0144] In some embodiments, the first member of the naturally occurring immunoglobulin super-family and its donor domain is identical in amino acid sequence to the second member of the naturally occurring immunoglobulin super-family and its donor domain respectively.

[0145] In some embodiments, the first member of the naturally occurring immunoglobulin super-family and its donor domain is different in amino acid sequence from the second member of the naturally occurring immunoglobulin super-family and its donor domain respectively.

[0146] In some embodiments the third and the fourth member of the naturally occurring immunoglobulin super-family, in particular the donor domains of the third and the fourth member of the naturally occurring immunoglobulin super-family, are different from the first and the second member of the naturally occurring immunoglobulin super-family, in particular the donor domains of the first and the second member of the naturally occurring immunoglobulin super-family. In some embodiments the first, second, third and fourth member of the naturally occurring immunoglobulin super-family, in particular its donor domains are each different from the other.

[0147] In some embodiments the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical. In some embodiments the first engineered immunoglobulin chain and its engineered domain are not identical to the second engineered immunoglobulin chain and its engineered domain, i.e. the engineered immunoglobulin chains, in particular its engineered domains are different in amino acid sequence from each other e.g. the first and the second engineered immunoglobulin chains in particular its engineered domains have amino acid sequences which are different from each other by at least one amino acid or by at least two amino acids or by at least three amino acids or by at least four amino acids or by at least five amino acids or by at least five to ten amino acids or by at least ten to thirty amino acids.

[0148] In some embodiments, the first engineered immunoglobulin chain and/or its engineered domain is different in amino acid sequence from the second engineered immunoglobulin chain and/or its engineered domain, wherein the protein-protein interfaces of the engineered domains of the engineered immunoglobulin chains interact by hetero-dimerization. In some embodiments, the first engineered immunoglobulin chain and/or its engineered domain is different in amino acid sequence from the second engineered immunoglobulin chain and/or its engineered domain, wherein the protein-protein interfaces of the engineered domains of the engineered immunoglobulin chains interact by homo-dimerization.

[0149] In some embodiments, at least one substituted amino acid residue in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is interacting with at least one substituted amino acid residue in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain. Preferably at least two, more preferably at least three, most preferably at least four, in particular at least five substituted amino acid residue in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain are interacting with at least one substituted amino acid residue in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain.

[0150] In some embodiments at least one, preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residue of the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain.

[0151] In some embodiments, each of the first and second engineered immunoglobulin chains and its engineered domains are different in amino acid sequence from the first and second member of the naturally occurring immunoglobulin super-family and its donor domains.

[0152] In some embodiments, the parent domain of the first engineered immunoglobulin chain is different in amino acid sequence from the first member of the naturally occurring immunoglobulin super-family and its donor domain. In some embodiments, the parent domain of the second engineered immunoglobulin chain is different in amino acid sequence from second member of the naturally occurring immunoglobulin super-family and its donor domain.

[0153] In some embodiments, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain are not substituted with charged amino acids, preferably are not substituted with a charged pair.

[0154] In some embodiments, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is not substituted with 26A and/or 26E and/or 86T and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is not substituted with 20T and/or 26K and/or 85.1A and/or 88I, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0155] In some embodiments, the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by homo-dimerization, wherein the engineered domains of the first engineered immunoglobulin chain and the second engineered immunoglobulin chain are not identical in amino acid sequence.

[0156] In some embodiments, the parent domains of the engineered domains of the engineered immunoglobulin chains are not substituted in the loop region of the domain.

[0157] In some embodiments, at least one 3D structural position of the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is different in amino acid residue compared to the identical 3D structural position of the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain.

[0158] In some embodiments, the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain are not identical to the third engineered immunoglobulin chain and its engineered domain, i.e. the engineered immunoglobulin chains, in particular its engineered domains are different in amino acid sequence from each other e.g. the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and/or the third engineered immunoglobulin chain, in particular the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and th engineered domain of the third engineered immunoglobulin chain have amino acid sequences which are different from each other by at least one amino acid or by at least two amino acids or by at least three amino acids or by at least four amino acids or by at least five amino acids or by at least five to ten amino acids or by at least ten to thirty amino acids.

[0159] In some embodiments, the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is different in amino acid sequence from the engineered domain of the third engineered immunoglobulin chain, wherein the protein-protein interfaces of the engineered domains of the engineered immunoglobulin chains interact by hetero-dimerization.

[0160] In some embodiments, the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is different in amino acid sequence from the engineered domain of the third engineered immunoglobulin chain, wherein the protein-protein interfaces of the engineered domains of the engineered immunoglobulin chains interact by homo-dimerization.

[0161] In some embodiments, the third member of the naturally occurring immunoglobulin super-family and its donor domain is different in amino acid sequence from the fourth member of the naturally occurring immunoglobulin super-family and its donor domain respectively.

[0162] In some embodiments, the third member of the naturally occurring immunoglobulin super-family and its donor domain is identical in amino acid sequence to the fourth member of the naturally occurring immunoglobulin super-family and its donor domain respectively.

[0163] In some embodiments, each of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain are different in amino acid sequence from the third and fourth member of the naturally occurring immunoglobulin super-family and its donor domains.

[0164] In some embodiments, the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is different in amino acid sequence from the third member of the naturally occurring immunoglobulin super-family and its donor domain.

[0165] In some embodiments, the parent domain of the engineered domain of the third engineered immunoglobulin chain is different in amino acid sequence from the fourth member of the naturally occurring immunoglobulin super-family and its donor domain.

[0166] In some embodiments the first and second engineered immunoglobulin chains have parent domains which are identical, in some embodiments the first and second engineered immunoglobulin chains have parent domains which are not identical. In some embodiments the first and second engineered immunoglobulin chains have parent domains which have amino acid sequences which are different from each other by one amino acid or by two amino acids or by three amino acids or by four amino acids or by five amino acids or by five to ten amino acids or by ten to thirty amino acids, preferably by at least one amino acid or by at least two amino acids or by at least three amino acids or by at least four amino acids or by at least five amino acids or by at least five to ten amino acids or by at least ten to thirty amino acids.

[0167] In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chains and the parent domain of the engineered domain of the third engineered immunoglobulin chain are identical. In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chains and the parent domain of the engineered domain of the third engineered immunoglobulin chain are not identical. In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chains and the parent domain of the engineered domain of the third engineered immunoglobulin chain are different from each other by one amino acid or by two amino acids or by three amino acids or by four amino acids or by five amino acids or by five to ten amino acids or by ten to thirty amino acids, preferably by at least one amino acid or by at least two amino acids or by at least three amino acids or by at least four amino acids or by at least five amino acids or by at least five to ten amino acids or by at least ten to thirty amino acids.

[0168] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof comprising a parent domain of the first and/or second engineered immunoglobulin chain, in particular a parent domain of the first and second engineered immunoglobulin chain which is selected from the group consisting of CH1, CH2, CH3, CH4, IGKC, IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7, CH1, CH3 can be from IGHA1, IGHA2, IGHD, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP and IGHM, C14 can be from IGHE and IGHM, CH2 can be from IGHA1, IGHA2, IGHD, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP and IGHM.

[0169] In some embodiments the parent domain of the first and/or the second engineered immunoglobulin chain, in particular the parent domain of the first and second engineered immunoglobulin chain, is a domain selected from the group consisting of CH1 domain, CH4 domain. IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain, preferably from the group consisting of CH domain, IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain.

[0170] Normally the parent domain of the first and/or second engineered immunoglobulin chain is a naturally occurring domain. Usually the parent domain of the first and/or second engineered immunoglobulin chain is from human e.g a naturally occurring human domain. Preferably the parent domain of the first and/or second engineered immunoglobulin chain is from human and from the same isotype, species and subclass. In some embodiments, the preferred parent domain of the first and/or second engineered immunoglobulin chain, in particular the parent domain of the first and second engineered immunoglobulin chain, is a CH3 domain, more preferably a human CH3 domain, in particular a human CH3 domain from IGHG1. Equally preferred, the parent domains of the first and second engineered immunoglobulin chain are from a different isotype. In some embodiments the parent domain of the first engineered immunoglobulin chain is a CH3 domain from IGHG1, preferably a human CH3 domain from IGHG1 and the parent domain of the second engineered immunoglobulin chain is a CH3 domain from IGHG3 preferably a human CH3 domain from IGIHG3.

[0171] In some embodiments, the parent domain of the first and/or second engineered immunoglobulin chain, in particular the parent domain of the first and second engineered immunoglobulin chain, is a CH4 domain, more preferably a human CH4 domain, in particular a human CH4 domain from IGHM or a human CH4 domain from IGHE.

[0172] Equally, the preferred the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain. Equally, the preferred parent domain of the first engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain and the preferred parent domain of the second engineered immunoglobulin chain is a CH1 domain. More preferred is a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1. Most preferably the parent domain of the first engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 or is an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain or the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 or is an IGKC domain.

[0173] In some embodiments the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain are selected from the group consisting of CH1, CH2, CH3, CH4, IGKC, IGLC1, IGLC2, IGLC3, IGLC6 and IGLC7. Normally the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a naturally occurring domain. Usually the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain is from human. Preferably the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain is from human and from the same isotyp, species and subclass. The preferred parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain is a CH3 domain, more preferably a human CH3 domain, in particular a human CH3 domain from IGHG1. Equally preferred, the parent domains of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and/or the engineered domain of the third engineered immunoglobulin chain are from a different isotype. In some embodiments the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is a CH3 domain from IGHG1, preferably a human CH3 domain from IGHG1, and the parent domain of the the engineered domain of the third engineered immunoglobulin chain is a CH3 domain from IGHG3 preferably a human CH3 domain from IGHG3.

[0174] In some embodiments, the parent domains of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and/or the engineered domain of the third engineered immunoglobulin chain is a CH4 domain, more preferably a human CH4 domain, in particular a human CH4 domain from IGHM or a human CH4 domain from IGHE. Equally preferred the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the the second engineered immunoglobulin is a CH1 domain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain. Equally, the preferred parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin is a light chain constant domain such as an IGLC domain or an IGKC domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is a CH1 domain. More preferred is a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1.

[0175] Parent domains from the same species, isotype and subclass, e.g. human CH3 domain from IGHG1 as parent domain of e.g. the first and second engineered immunoglobulin chain, form a homo-dimer. In some embodiments the parent domains of the first and the second engineered immunoglobulin chain form a homo-dimer, specifically a naturally occurring homo-dimer.

[0176] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain form a hetero-dimer specifically a naturally occurring hetero-dimer, e.g. a CH1 domain and an IGKC domain or a CH1 domain and an IGLC domain.

[0177] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain form a hetero-dimer e.g. a CH3 domain from IGHG1 and a CH3 domain from IGHG3.

[0178] In some embodiments the parent domains of the further engineered domain of the first and/or second engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain form a homo-dimer, specifically a naturally occurring homo-dimer.

[0179] In some embodiments the parent domains of the further engineered domain of the first and/or second engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain form a hetero-dimer, specifically a naturally occurring hetero-dimer, e.g. a CH1 domain and an IGKC domain or a CH1 domain and an IGLC domain.

[0180] In some embodiments the parent domains of the further engineered domain of the first and/or second engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain form a hetero-dimer e.g. a CH3 domain from IGHG1 and a CH3 domain from IGHG3.

[0181] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain.

[0182] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain are not a CH3 domain.

[0183] In some embodiments the parent domains of the first and the second engineered immunoglobulin chains are not a VH domain and not a VL domain.

[0184] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain are not from IgG2.

[0185] In some embodiments the preferred parent domain of the first engineered immunoglobulin chain is a CH1 domain and the preferred parent domain of the second engineered immunoglobulin chain is an IGKC domain or, the preferred parent domain of the first engineered immunoglobulin chain is an IGKC domain and the preferred parent domain of the second engineered immunoglobulin chain is a CH1 domain. Even more preferably, the preferred parent domain of the first engineered immunoglobulin chain is a human CH1 domain and the preferred parent domain of the second engineered immunoglobulin chain is a human IGKC domain or, the preferred parent domain of the first engineered immunoglobulin chain is a human IGKC domain and the preferred parent domain of the second engineered immunoglobulin chain is a human CH1 domain, more preferably a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1.

[0186] In some embodiments the preferred parent domain of the first engineered immunoglobulin chain is a CH1 domain and the preferred parent domain of the second engineered immunoglobulin chain is an IGLC domain or, the preferred parent domain of the first engineered immunoglobulin chain is an IGLC domain and the preferred parent domain of the second engineered immunoglobulin chain is a CH1 domain. Even more preferably, the preferred parent domain of the first engineered immunoglobulin chain is a human CH1 domain and the preferred the parent domain of the second engineered immunoglobulin chain is a human IGLC domain or, the preferred parent domain of the first engineered immunoglobulin chain is a human IGLC domain and the preferred parent domain of the second engineered immunoglobulin chain is a human CH1 domain, more preferably a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1.

[0187] In some embodiments the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain or, the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is an IGKC domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain. Even more preferably, the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a human CH1 domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is a human IGKC domain or, the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a human IGKC domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is a human CH1 domain, more preferably a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1.

[0188] In some embodiments the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGLC domain or, the preferred parent domain of the further engineered domain, of the first and/or second engineered immunoglobulin chain is an IGLC domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain. Even more preferably, the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a human CH1 domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is a human IGLC domain or, the preferred parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a human IGLC domain and the preferred parent domain of the engineered domain of the third engineered immunoglobulin chain is a human CH1 domain. More preferred is a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1.

[0189] In some embodiments the parent domain of the first and/or second engineered immunoglobulin chain, in particular the parent domain of the first and second engineered immunoglobulin chain, is a CH3 domain, preferably a CH3 domain of the same species, isotype and subclass, and the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin is a tight chain constant domain such as an IGLC domain or an IGKC domain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a CH1 domain.

[0190] In some embodiments the parent domain of the first engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain and the parent domain of the engineered domain of the second engineered immunoglobulin chain is a CH1 domain, and the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a CH3 domain, preferably a CH3 domain of the same species, isotype and subclass.

[0191] In some embodiments e.g if the hetero-dimeric immunoglobulin is a F(ab').sub.2, the parent domain of the engineered domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the engineered domain of the second engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain and the parent domain of the further engineered domain of the first engineered immunoglobulin chain is a CH1 domain, and the parent domain of the further engineered domain of the second engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain, wherein the first engineered immunoglobulin chain comprises a hinge region between the parent domain of the engineered domain and the parent domain of the further engineered domain.

[0192] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the amino acid residues from the protein-protein interface of the donor domain of the first and second member of the naturally occurring immunoglobulin super-family are amino acids non essential to the core integrity of the domain, preferably wherein the amino acid residues from the protein-protein interface of the donor domain of the first and second member of the naturally occurring immunoglobulin super-family used for substitution of the protein-protein interface of the parent domain of the first and/or second engineered immunoglobulin chain are amino acids non essential to the core integrity of the domain.

[0193] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, and 88 or comprises an amino acid residue substitution at a position selected from the group consisting of 85.1 and 86, more preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1 and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, and 88 or comprises an amino acid residue substitution at a position selected from the group consisting of 85.1 and 86, in particular the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at position 88 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, and 88 or comprises an amino acid residue substitution at a position selected from the group consisting of 85.1 and 86, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0194] Thus in some embodiments, the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, 88 and/or, wherein the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, 88, wherein if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, preferably not charged amino acids, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0195] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at position 20 selected from the group consisting of 20A, 20E, 20K, 20N, 20Q, 20S, 20T, 20V and 20W and/or conservative amino substitutions thereof, preferably selected from the group consisting of 20A, 20N, 20Q, 20S, 20T, 20V and 20W and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0196] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at position 22 selected from the group consisting of 22A, 22G, 22I, 22L, 22T and 22V and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0197] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at position 85.1 selected from the group consisting of 85.1A, 85.1C, 85.1F, 85.1H, 85.1K, 85.1M, 85.1N, 85.1R, 85.1R, 85.1T, and 85.1W and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0198] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at position 86 selected from the group consisting of 86F, 86H, 86I, 86T, 86Q, 86S, 86V, 86W, 86Y and/or conservative amino substitutions thereof; wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0199] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at position 88 selected from the group consisting of 88E, 88I, 88K, 88L, 88Q, 88R, 88T, 88V, 88W, 88Y and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0200] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at position 22 selected from the group consisting of 22A, 22G, 22I, 22L, 22T and 22V and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0201] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at position 26 selected from the group consisting of 26K, 26Q, 26R, 26S, 26T, and 26V and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0202] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at position 85.1 selected from the group consisting of 85.1A, 85.1C, 85.1F, 85.1F, 85.1K, 85.1M, 85.1N, 85.1R, 85.1S, 85.1T, and 85.1W and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0203] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at position 86 selected from the group consisting of 86F, 86H, 86I, 86T, 86Q, 86S, 86V, 86W, 86Y and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0204] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at position 88 selected from the group consisting of 88E, 88I, 88K, 88L, 88Q, 88R, 88T, 88V, 88W, 88Y and/or conservative amino substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0205] In some embodiments, if the substitution in the parent domain of the first engineered immunoglobulin chain is at position 88, the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 88 in the parent domain of the first engineered immunoglobulin chain is at position 85.1 and/or 86, or if the substitution in the parent domain of the second engineered immunoglobulin chain is at position 88, the substituted amino acid residue in the patent domain of the first engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 88 in the parent domain of the second engineered immunoglobulin chain is at position 85.1 and/or 86, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0206] In some embodiments, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at position 88 and the substituted amino acid residue in the patent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 85.1 and/or 86, preferably at position 85.1 and 86, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. Usually the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain at position 88 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain at position 85.1 and/or 86 interact with each other, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0207] In some embodiments, if the substitution in the parent domain of the first engineered immunoglobulin chain is at position 85.1, the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 85.1 in the parent domain of the first engineered immunoglobulin chain is at position 86, or if the substitution in the parent domain of the second engineered immunoglobulin chain is at position 85.1, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 85.1 in the parent domain of the second engineered immunoglobulin chain is at position 86, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0208] In some embodiments, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at position 85.1 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 86, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. Usually the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain at position 85.1 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain at position 86 interact with each other, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0209] In some embodiments, if the substitution in the parent domain of the first engineered immunoglobulin chain is at position 22, the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 22 in the parent domain of the first engineered immunoglobulin chain is at position 22 and/or 86, or if the substitution in the parent domain of the second engineered immunoglobulin chain is at position 22, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 22 in the parent domain of the second engineered immunoglobulin chain is at position 22 and/or 86, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0210] In some embodiments, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at position 22 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 22 and/or 86, preferably at position 22 and 86, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. Usually the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain at position 22 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain at position 22 and/or 86 interact with each other, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0211] In some embodiments, if the substitution in the parent domain of the first engineered immunoglobulin chain is at position 20, the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 20 in the parent domain of the first engineered immunoglobulin chain is at position 26, or if the substitution in the parent domain of the second engineered immunoglobulin chain is at position 20, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain which interacts with the substituted amino acid residue at position 20 in the parent domain of the second engineered immunoglobulin chain is at position 26, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0212] In some embodiments, the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at position 20 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 26, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. Usually the substituted amino acid residue in the parent domain of the first engineered immunoglobulin chain at position 20 and the substituted amino acid residue in the parent domain of the second engineered immunoglobulin chain at position 26 interact with each other, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0213] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHA1 CH2 or a human IGHA2 CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0214] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHA1 CH2 or a human IGHA2 CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof; and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0215] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHA1 CH2 or a human IGHA2 CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0216] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHD CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88R and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86H and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0217] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHD CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86H and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof; wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0218] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHD CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86H and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0219] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88E and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86Q and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0220] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0221] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22L and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0222] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG1 CH2 or a human IGHG2 CH2 or a human IGHG3 CH2 or a human IGHG4 CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and/or 85.1R and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0223] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG1 CH2 or a human IGHG2 CH2 or a human IGHG3 CH2 or a human IGHG4 CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1R and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0224] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG1. CH2 or a human IGHG2 CH2 or a human IGHG3 CH2 or a human IGHG4 CH2 wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof; wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0225] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHP CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and/or 85.1H and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0226] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHP CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1H and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHP CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0227] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and/or 85.1K and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0228] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1K and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0229] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22I and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0230] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHA1 CH3 or a human IGHA2 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88I and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHA1 CH3 or a human IGHA2 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0231] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHA1 CH3 or a human IGHA2 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0232] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHD CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86W and conservative amino acid substitutions thereof, and/or 85.1W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0233] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHD CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86W and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0234] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin superfamily are a human IGHD CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86W and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22L and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof; and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and/or 85.1T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0235] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0236] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG1 CH3 or a human IGHG2 CH3 or a human IGHG3 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88K and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof, and/or 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0237] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG1 CH3 or a human IGHG2 CH3 or a human IGHG3 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof; and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0238] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG1 CH3 or a human IGHG2 CH3 or a human IGHG3 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0239] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG4 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88R and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof and/or 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0240] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG4 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0241] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHG4 CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHP CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88K and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof, and/or 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0242] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHP CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0243] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHP CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0244] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88E and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0245] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH3, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0246] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH4, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88R and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof, and/or 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0247] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH4, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHE CH4, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0248] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH4, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88I and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86H and conservative amino acid substitutions thereof, and/or 85.1F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0249] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH4, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86H and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1F and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0250] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGHM CH4, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0251] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0252] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0253] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0254] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0255] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0256] In some embodiments, the donor domains of the first and second member of the naturally occurring immunoglobulin super-family are a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0257] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or human IGHA2 CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or human IGHA2 CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0258] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or human IGHA2 CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0259] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0260] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0261] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88L and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0262] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86I and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0263] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein Interface of the engineered domain of the first immunoglobulin chain comprise 86I and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0264] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0265] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0266] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGH4 CH1 or a human IGHGP CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0267] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0268] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0269] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGKC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof; wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0270] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Q and conservative amino acid substitutions thereof; and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0271] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0272] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0273] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0274] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0275] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0276] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88L and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0277] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, and the donor domain of the second member of the naturally occurring immunoglobulin superfamily is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86I and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86I and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. 8 numbering.

[0278] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0279] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof; wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGLC2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0280] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Q and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0281] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0282] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0283] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0284] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0285] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0286] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0287] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0288] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, wherein the amino acid substitutions in the protein-protein Interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof; and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86I and conservative amino acid substitutions thereof; and/or 85.1A and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0289] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0290] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22G and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0291] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGIHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0292] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22G and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0293] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88T and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0294] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGKC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22G and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0295] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof and/or 85.1T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0296] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1T and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0297] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHA1 CH1 or a human IGHA2 CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0298] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGC12 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0299] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHD CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0300] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86I and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0301] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0302] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHE CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22G and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0303] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0304] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGHG2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0305] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHG1 CH1 or a human IGLC2 CH1 or a human IGHG3 CH1 or a human IGHG4 CH1 or a human IGHGP CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22G and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0306] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHCM CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88Y and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86T and conservative amino acid substitutions thereof, and/or 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0307] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0308] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human IGLC1 or a human IGLC2 or a human IGLC3 or a human IGLC6 or a human IGLC7, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human IGHM CH1, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22G and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0309] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRAC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRBC1 or a human TRBC2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88W and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and/or 85.1C or 85.1A or 85.1C and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0310] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRAC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRBC1 or a human TRBC2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1C or 85.1A or 85.1C and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0311] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRAC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRBC1 or a human TRBC2, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0312] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRBC1 or a human TRBC2, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRAC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88R and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof and/or 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0313] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRBC1 or a human TRBC2, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRAC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0314] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRBC1 or a human TRBC2, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRAC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86S and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRDC, and the donor domain of the second member of the naturally occurring immunoglobulin super family is a human TRGC1 or a human TRGC2 (2.times.) or a human TRGC2 (3.times.), wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88L and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof and/or 85.1M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0315] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRDC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRGC1 or a human TRGC2 (2.times.) or a human TRGC2 (3.times.), wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRDC, and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRGC1 or a human TRGC2 (2.times.) or a human TRGC2 (3.times.), wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22L and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0316] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRGC1 or a human TRGC2 (2.times.) or a human TRGC2 (3.times.), and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRDC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 88W and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 86V and conservative amino acid substitutions thereof, and/or 85.1N and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRGC1 or a human TRGC2 (2.times.) or a human TRGC2 (3.times.), and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRDC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 85.1N and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0317] In some embodiments, the donor domain of the first member of the naturally occurring immunoglobulin super-family is a human TRGC1 or a human TRGC2 (2.times.) or a human TRGC2 (3.times.), and the donor domain of the second member of the naturally occurring immunoglobulin super-family is a human TRDC, wherein the amino acid substitutions in the protein-protein interface of the engineered domain of the first immunoglobulin chain comprise 86F and conservative amino acid substitutions thereof, and the amino acid substitutions in the protein-protein interface of the engineered domain of the second immunoglobulin chain comprise 22A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0318] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20E, 20K, 20W, 20S, 22A, 22G, 22T, 22L, 22I, 22V, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1W, 85.1P, 85.1C, 85.1N, 86S, 86I, 86T, 86H, 86Q, 86V, 86W, 86Y, 86F, 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88K, 88Y, and 88W and/or wherein the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution selected from the group consisting of 22A, 22G, 22T, 22L, 22I, 22V, 26Q, 26T, 26K, 26V, 26S, 26R, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1W, 85.1F, 85.1C, 85.1N, 86S, 86I, 86T, 86H, 86Q, 86V, 86W, 86Y, 86F, 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88K, 88Y, and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0319] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution selected from the group consisting of 20K, 20N, 20T, 20S, 22A, 22L, 22V, 22T, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86F, 86Y, 86V, 88W, 88R, 88L, and 88K and/or conservative amino acid substitutions thereof and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution selected from the group consisting of 22A, 22L, 22V, 22T, 26E, 26I, 26K, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86F, 86Y, 86V, 88W, 88R, 88L, and 88K and/or conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0320] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution selected from the group consisting of 20K, 20N, 20T, 20S, 22A, 22L, 22V, 22T, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86T, 86F, 86Y, 86V, 88W, 88R, 88I, 88T, and 88K and/or conservative amino acid substitutions thereof and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution selected from the group consisting of 22A, 22L, 22V, 22T, 26E, 26T, 26K, 26R, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86T, 86F, 86Y, 86V, 88W, 88R, 88L, 88I and 88K and/or conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0321] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises a further amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 26, 27, 79, 81, 83, 84, 84.2 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0322] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises a further amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 20, 27, 79, 81, 83, 84, 84.2 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0323] Preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, 88 and a further amino acid residue substitution at a position selected from the group consisting of 26, 79, and 90, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, 88 and a further amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 20, 81, 84, 84.2, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0324] Preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, 88 and a further amino acid residue substitution at a position selected from the group consisting of 3, 5, 26, 27, 81, 83, and 84, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 85.1, 86, 88 and a further amino acid residue substitution at a position selected from the group consisting of 7, 20, 27, 79, 81, 84.2, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0325] Preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, 88 and a further amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 26, 79, 81, 83, and 84.2, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, 88 and a further amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 20, 79, 81, 83, and 84.2, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0326] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the parent domain of the first and/or the second engineered immunoglobulin chain is a domain selected from the group consisting of CH1 domain, CH4 domain. IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0327] Preferably, the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain. Equally preferably the parent domain of the first and the second engineered immunoglobulin chain is a CH4 domain, in particular a CH4 domain of the same isotype, species and subclass.

[0328] Preferably, the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGKC domain or, the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain.

[0329] Preferably, the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 or, the parent domain of the first engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 and the parent domain of the second engineered immunoglobulin chain is a CH1 domain.

[0330] In some embodiments, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, the parent domain of the first and/or the second engineered immunoglobulin chain is a domain selected from the group consisting of CH1 domain, CH4 domain, IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0331] In some embodiments, wherein the parent domains of the first and the second engineered immunoglobulin chain are not a CH2 domain, not a CH3 domain, not a VL domain and not a VH domain and wherein,

if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 12 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 119, the substituted amino acid residue at position 12 and the substituted amino acid residue at position 119 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 26 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 13, the substituted amino acid residue at position 26 and the substituted amino acid residue at position 13 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 5 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 20, the substituted amino acid residue at position 5 and the substituted amino acid residue at position 20 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGLC domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 27 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 18, the substituted amino acid residue at position 27 and the substituted amino acid residue at position 18 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 20 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 26, the substituted amino acid residue at position 20 and the substituted amino acid residue at position 26 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 79 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 84.3, the substituted amino acid residue at position 79 and the substituted amino acid residue at position 84.3 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGLC domain or an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is substituted with an amino acid residue at position 86 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is substituted with an amino acid residue at position 86, the substituted amino acid residues at both positions 86 are not a charged pair, if the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at position 11 and the parent domain of the second engineered immunoglobulin chain is a domain which comprises a hinge region, the parent domain of the second engineered immunoglobulin chain is not substituted at position 3 of the hinge region, the parent domain of the first and/or the second engineered immunoglobulin chain is a domain selected from the group consisting of CH1 domain, CH4 domain, IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain and/or the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first and/or the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 85.1, 86, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0332] Preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 26, 79, 85.1, 86, 88 and 90 and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 85.1, 86, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0333] Preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 3, 5, 20, 22, 26, 27, 81, 83, 84, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 85.1, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0334] Preferably the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 20, 22, 26, 79, 81, 83, 84.2, 85.1, 86, and 88, and the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 3, 5, 7, 11, 12, 13, 18, 20, 22, 26, 79, 81, 83, 84.2, 83.1, 86, and 88, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0335] In some embodiments the first and the second member of the naturally occurring immunoglobulin super-family are selected from the TCR constant domain family. In some embodiments the third and the fourth member of the naturally occurring immunoglobulin super-family are selected from the TCR constant domain family.

[0336] Preferably the donor domain of first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2). Equally the donor domain of the first member of the naturally occurring immunoglobulin super-family can be the TCR constant domain beta (SEQ ID NO: 2) and the donor domain of the second member of the naturally occurring immunoglobulin super-family can be the TCR constant domain alpha (SEQ ID NO: 1).

[0337] Preferably the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2). Equally the donor domain of the third member of the naturally occurring immunoglobulin super-family can be the TCR constant domain beta (SEQ ID NO: 2) and the donor domain of the fouth member of the naturally occurring immunoglobulin super-family can be the TCR constant domain alpha (SEQ ID NO: 1).

[0338] In a further embodiment the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2), wherein the cysteine (C) at amino acid position 75 in SEQ ID NO: 2 is substituted with alanine (A) or serine (S), preferably with alanine (A).

[0339] In a further embodiment the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2), wherein the cysteine (C) at amino acid position 75 in SEQ ID NO: 2 is substituted with alanine (A) or serine (S), preferably with alanine (A).

[0340] In a further embodiment the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) or wherein the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0341] In a further embodiment the donor domain of the first and/or second member of the naturally occurring immunoglobulin super-family is an IgA1 CH3 domain, preferably an IGHA1 CH3 domain, more preferably the human IGHA1 CH3 domain (SEQ ID NO: 96). In a further embodiment the donor domain of the first and/or second member of the naturally occurring immunoglobulin super-family is IgA2 CH3 domain, preferably an IGHA2 CH3 domain, more preferably the human IGHA2 CH3 domain (SEQ ID NO: 97).

[0342] In a further embodiment the donor domain of the third and/or fourth member of the naturally occurring immunoglobulin super-family is an IgA1 CH3 domain, preferably an IGHA1 CH3 domain, more preferably the human IGHA1 CH3 domain (SEQ ID NO: 96). In a further embodiment the donor domain of the third and/or fourth member of the naturally occurring immunoglobulin super-family is an IgA2 CH3 domain, preferably an IGHA2 CH3 domain, more preferably the human IGHA2 CH3 domain (SEQ ID NO: 97).

[0343] The IGHA1 CH3 domain or the IGHA2 CH3 domain as donor domains are particularly useful if the parent domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain or the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain are CH3 domains from an isotype different from IGHA1 or IGHA2, e.g. IGHG1 or IGHG2 or IGHG3 or IGHG4 CH3 domains or preferably IGHG1 CH3, more preferably human IGHG1 CH3.

[0344] In a further embodiment at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family and additionally at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a third member of the naturally occurring immunoglobulin super-family and wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family and additionally at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a fourth member of the naturally occurring immunoglobulin super-family.

[0345] In case the parent domains are from IgG1, the donor domain of the first member of the naturally occurring immunoglobulin super-family can be selected from e.g. a CH3 domain of IgA, IgM or IgE, the donor domain of the third member of the naturally occurring immunoglobulin super-family can be selected from e.g. a CH4 domain of IgM or IgE or from a CH3 domain of IgG2, IgG3 or IgG4, the donor domain of the second member of the naturally occurring immunoglobulin super-family can be selected from e.g. a CH3 domain of IgA, IgM or IgE and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family can be selected from e.g. a CH4 domain of IgM or IgE or from a CH3 domain of IgG2, IgG3 or IgG4. Usually the donor domain of the first member of the naturally occurring immunoglobulin super-family and the the donor domain of the second member of the naturally occurring immunoglobulin super-family which are selected are the same and the donor domain of the third member of the naturally occurring immunoglobulin super-family and the the donor domain of the fourth member of the naturally occurring immunoglobulin super-family are the same.

[0346] Provided that the parent domain of the first engineered immunoglobulin chain and the parent domain of the second engineered immunoglobulin chain form a hetero-dimer, specifically a naturally occurring hetero-dimer, or are specially a CH1 domain and an IGKC domain, the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin can be domains which form a homo-dimer, usually domains of the same isotype, species and subclass, preferably a CH3 domain, more preferred a human CH3 domain, even more preferred an IgG1 CH3 domain, in particular a human IgG1 CH3 domain. Equally preferred is an IgA1 CH3 domain or an IgA2 CH3 domain, preferably the human IGHA1 CH3 domain or the human IGHA2 CH3 domain.

[0347] Provided that the parent domain of the first engineered immunoglobulin chain and the parent domain of the second engineered immunoglobulin chain form a hetero-dimer, specifically a naturally occurring hetero-dimer, or are specifically a CH1 domain and an IGKC domain, the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin can be domains which form a hetero-dimer, preferably domains from the TCR constant domain family, more preferably TCR constant domain alpha (SEQ ID NO: 1) and TCR constant domain beta (SEQ ID NO: 2) or TCR constant domain gamma (SEQ ID NO: 33) and the TCR constant domain delta (SEQ ID NO: 32).

[0348] Provided that the parent domain of the first engineered immunoglobulin chain and the parent domain of the second engineered immunoglobulin chain form a homo-dimer, specifically a naturally occurring homo-dimer, or are specifically domains of the same isotype, species and subclass, the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin are domains which form a heterodimer, preferably domains from the TCR constant domain family, more preferably TCR constant domain alpha (SEQ ID NO: 1) and TCR constant domain beta (SEQ ID NO: 2) or TCR constant domain gamma (SEQ ID NO: 33) and the TCR constant domain delta (SEQ ID NO: 32).

[0349] Provided that the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain form a hetero-dimer, specifically a naturally occurring hetero-dimer, or are specifically a CH1 domain and a IGKC domain, the donor domain of the third member of the naturally occurring immunoglobulin super-family and the donor domain of the fourth member of the naturally occurring immunoglobulin are preferably domains which form a homo-dimer, usually domains of the same isotype, species and subclass, preferably a CH3 domain, more preferred a human CH3 domain, even more preferred an IgG1 CH3 domain, in particular a human IgG1 CH3 domain.

[0350] Provided that the parent domain of the further engineered domain of the first and/or second engineered Immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain form a homo-dimer, specifically a naturally occurring homo-dimer, or are specifically domains of the same isotype, species and subclass, the donor domain of the third member of the naturally occurring immunoglobulin super-family and the donor domain of the fourth member of the naturally occurring immunoglobulin are preferably domains which form a hetero-dimer, preferably domains from the TCR constant domain family, more preferably TCR constant domain alpha (SEQ ID NO: 1) and TCR constant domain beta (SEQ ID NO: 2) or TCR constant domain gamma (SEQ ID NO: 33) and the TCR constant domain delta (SEQ ID NO: 32).

[0351] Provided that the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain form a hetero-dimer, specifically a naturally occurring hetero-dimer, the donor domain of the third member of the naturally occurring immunoglobulin super-family and the donor domain of the fourth member of the naturally occurring immunoglobulin are domains which form a hetero-dimer, preferably domains from the TCR constant domain family, more preferably TCR constant domain alpha (SEQ ID NO: 1) and TCR constant domain beta (SEQ ID NO: 2) or TCR constant domain gamma (SEQ ID NO: 33) and the TCR constant domain delta (SEQ ID NO: 32).

[0352] In some embodiments the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2), the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0353] In some embodiments the the donor domain of first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33), the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2).

[0354] In some embodiments the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) or the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2) or the TCR constant domain gamma (SEQ ID NO: 33), the donor domain of the third and the fourth member of the naturally occurring immunoglobulin super-family is the IgG1 CH3 domain (SEQ ID NO: 47), with the proviso that if the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2) and if the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0355] In some embodiments the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) or the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2) or the TCR constant domain gamma (SEQ ID NO: 33), the donor domain of the third and the fourth member of the naturally occurring immunoglobulin super-family is the human IGHA1 CH3 domain (SEQ ID NO: 96) or the human IGHA2 CH3 domain (SEQ ID NO: 97), with the proviso that if the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: I), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2) and if the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0356] In some embodiments the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by hetero-dimerization.

[0357] In some embodiments the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by homo-dimerization.

[0358] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 118, 123, 124, 131, 133, 137, 138, 160, 162, 164, 165, 167, 174, 176, 178, and 180, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0359] In some embodiments the parent domain of the first engineered Immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 118, 122, 131, 133, 137, 138, 160, 162, 165, 167, 174, 176, 178, and 180, wherein the amino acid position of each group member is indicated according to the Kabat numbering.

[0360] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S131V, S131T, S131A, S131N, S13Q, S131E, S131K, S131W, V133A, V133G, V133T, V133L, V133I, N137Q, N137T, N137K, N137V, N137S, N137R, N137E, S174T, S174M, S174A, S174R, S174H, S174K, S174W, S174F, S174C, S174N, S176I, S176T, S176H, S176Q, S176V, S176W, S176Y, S176F, T178Q, T178L, T178V, T178R, T178E, T178I, T178K, T178Y, and T178W, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0361] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114E, S114K, S114Q, F116A, F116T, F116Y, F118M, F118L, E123D, Q124E, S131K, S131T, S131N, V133L, V133A, V133T, N137T, N137E, N137K, N138K, N138E, Q160Y, Q160F, Q160K, S162A, S162, S1620, S162T, T164V, T164M, E165L, D167E, D167S, S174A, S174M, S174N, S174F, S176F, S176V, S176Y, T178W, T178R, T178L, T178K, T180N, T180R, and T180K or is selected from the group consisting of S114, S114K, S114Q, F116A, F116T, F116Y, F118M, F118L, E123D, Q124E, S131K, S131T, S131N, V133L, V133A, V133T, N137T, N137E, N137K, N138K, N138E, Q160Y, Q160F, Q160K, S162A, S162G, S162D, S162T, T164V, T164M, E165L, D167E, D167S, S174A, S174M, S174N, S174F, S176F, S176V, S176Y, T178W, T178R, T178L, T178K, T180N, T180R, and T180K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0362] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of 131V, 131A, 131N, 131Q, 131E, 131K, 131W, 131S, 133A, 133G, 133T, 133L, 133I, 137Q, 137T, 137K, 137V, 137R, 137N, 137E, 174T, 174M, 174S, 174R, 174H, 174K, 174W, 174F, 174C, 174N, 176I, 176I; 176H, 176Q, 176W, 176Y, 176V, 176F, 178Q, 178L, 178V, 178R, 178F, 178T, 178I, 178K, 178E, and 178W, wherein the amino acid position of each group member is indicated according to the Kabat numbering.

[0363] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of 114E, 114K, 114Q, 116A, 116Y, 118M, 18L, 122D, 131K, 131N, 133A, 133L, 133T, 137T, 137E, 137K, 138E, 138K, 160Y, 160F, 160K, 162D, 162A, 162G, 165L, 165M, 167E, 167S, 174N, 174M, 174F, 174S, 176V, 176F, 176Y, 178W, 178R, 178L, 178K, 180N, 180R, and 180K or selected from the group consisting of 114E, 114K, 114Q, 116A, 116Y, 118M, 118L, 122D, 131K, 131N, 133A, 133L, 133T, 137T, 137E, 137K, 138E, 138K, 160Y, 160F, 160K, 162D, 162A, 162G, 1651, 165M, 167E, 167S, 174N, 174M, 174F, 174S, 176V, 176F, 176Y, 178W, 178R, 178L, 178K, 180N, 180R, and 180K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the Kabat numbering.

[0364] In some embodiments the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 128, 133, 134, 139, 141, 143, 147, 148, 168, 170, 173, 175, 181, 183, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0365] In some embodiments the parent domain of the second engineered immunoglobulin chain is a CH1 domain and and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of 141V, 141T, 141N, 141Q, 141E, 141K, 141W, 141S, 143A, 143T, 143L, 143I, 143V, 147Q, 147T, 147V, 147S, 147R, 147N, 147E, 181T, 181M, 181A, 181R, 181H, 181K, 181W, 181F, 181C, 181N, 183I, 183H, 183Q, 183V, 183T, 183W, 183Y, 183F, 185Q, 185L, 185R, 185E, 185I, 185T, 185K, 185Y, and 185W, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0366] In some embodiments the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124E, S124K, S124Q, F126A, F126T, P126Y, L128F, L128M, K133D, S134E, T139Q, A141K, A141T, A141N, A141S, G143V, G143A, G143L, G143T, K147T, K147E, D148E, D148K, H168Y, H168F, H168K, F170D, F170A, F170G, F170T, V173L, V173M, Q175E, Q175S, Q175D, S181A, S181N, S181M, S181F, S183V, S183F, S183Y, V185W, V185R, V185L, V185K, T187N, T187K and T187R or is selected from the group consisting of S124E, S124K, S124Q, F126A, F126T, F126Y, L128F, L128M, K133D, S134E, T139Q, A141K, A141T, A141N, A141S, G143V, G143A, G143L, G143, K147T, K147E, D148E, D148K, H168Y, H168F, H168K, F170D, F170A, F170G, F170T, V173L, V173M, Q175E, Q175S, Q175D, S181A, S181N, S181M, S181F, S183V, S183F, S183Y, V185W, V185R, V185L, V185K, T187N, T187K and T187R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0367] Positions 114, 116, 118, 122, 131, 133, 137, 138, 160, 162, 165, 167, 174, 176, 178, and 180 in the protein-protein interface of an IGLC domain as parent domain according to Kabat numbering correspond in the same order to positions 3, 5, 7, 11, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88, and 90 indicated according to the IMGT.RTM. numbering.

[0368] Positions 114, 116, 118, 123, 124, 131, 133, 137, 138, 160, 162, 164, 165, 167, 174, 176, 178, and 180 in the protein-protein interface of the IGKC domain as parent domain according to EU numbering correspond in the same order to positions 3, 5, 7, 12, 13, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 85.1, 86, 88, and 90 indicated according to the IMGT.RTM. numbering.

[0369] Positions 124, 126, 128, 133, 134, 139, 141, 143, 147, 148, 168, 170, 173, 175, 181, 183, 185, and 187 in the protein-protein interface of the CH1 domain as parent domain according to EU numbering correspond in the same order to positions 3, 5, 7, 12, 13, 18, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88, and 90 indicated according to the IMGT.RTM. numbering.

[0370] Positions 124, 126, 128, 133, 134, 139, 141, 143, 147, 148, 168, 170, 173, 175, 181, 183, 185, and 187 in the protein-protein interface of the CH4 domain as parent domain according to EU numbering correspond in the same order to positions 3, 5, 7, 12, 13, 18, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88, and 90 indicated according to the IMGT.RTM. numbering.

[0371] Thus equivalently in some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the engineered domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 3, 5, 7, 12, 13, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 85.1, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0372] Thus equivalently in some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the first engineered immunoglobulin chain is an IGLC domain selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 3, 5, 7, 11, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0373] Thus equivalently in some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the engineered domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 3, 5, 7, 12, 13, 18, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0374] Thus equivalently in some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the engineered domain of the first and/or second engineered immunoglobulin chain is a CH4 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 3, 5, 7, 12, 13, 18, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0375] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 131, 137, 160, 176, 178, and 180, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 128, 141, 143, 147, 170, 173, 175, 181, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0376] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 178 or comprises T178W or comprises T178W and conservative amino acid substitutions thereof, and optionally a further amino acid residue selected from the group consisting of 131, 137, 160, 176, and 180, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 181 or comprises S181A or comprises S181A and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 124, 126, 128, 141, 143, 147, 170, 173, 175, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0377] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S131K, N137T, Q160Y, S176V, T178W, and T180N or comprises an amino acid residue selected from the group consisting of S131K, N137T, Q160Y, S176V, T178W, and T180N and conservative amino acid substitutions thereof, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124E, F126A, L128F, A141T, G143V, K147T, F170D, V173L, Q175E, S181A, V185R or comprises an amino acid residue selected from the group consisting of S124E, F126A, L128F, A141T, G143V, K147T, F170D, V173L, Q175E, S181A, V185R and T187R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0378] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 131, 137, 162, 165, 167, 174, 178, and 180, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 141, 143, 147, 168, 183, 185 and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0379] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at position 174 or comprises S174A or comprises S174A and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 114, 116, 131, 137, 162, 165, 167, 178, and 180, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 185 or comprises V185W or comprises V185W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 141, 143, 147, 168, 183, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0380] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114E, F116A, S131T, N137T, S162D, E165L, D167E, S174A, T178R, and T180R or comprises an amino acid residue selected from the group consisting of S114E, F116A, S131T, N137T, S162D, E165L, D167E, S174A, T178R, and T180R and conservative amino acid substitutions thereof, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of A141K, G143V, K147T, H168Y, S183V, V185W, and T187N or comprises an amino acid residue selected from the group consisting of A141K, G143V, K147T, H168Y, S183V, V185W, and T187N and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0381] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 131, 133, 137, 138, 162, 164, 174, 176, 178 and 180, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 128, 141, 143, 148, 168, 170, 175, 181, 183, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0382] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 178 or comprises T178W or comprises T178W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 114, 116, 131, 133, 137, 138, 162, 164, 174, and 176, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at position 183 or comprises S183V or comprises S183V and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 128, 141, 143, 148, 168, 170, 175, 181, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0383] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114K, F116T, S131T, V133L, N137E, N138K, S162G, T164M, S174M, S176F, and T178W or comprises an amino acid residue selected from the group consisting of S114K, F116T, S131T, V133L, N137E, N138K, S162G, T164M, S174M, S176F, and T178W and conservative amino acid substitutions thereof, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of L128M, A141N, G143A, D148E, H168F, F170A, Q175S, S181N, S183V, V185L, and T187K or comprises an amino acid residue selected from the group consisting of L128M, A141N, G143A, D148E, H168F, F170A, Q175S, S181N, S183V, V185L, and T187K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0384] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 118, 131, 133, 138, 160, 162, 167, 174, 176, 178, and 180 and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 141, 143, 147, 148, 170, 173, 181, 183 and 185, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0385] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 176 or comprises S176V or comprises S176V and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 118, 131, 133, 138, 160, 162, 167, 174, 178, 180, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 185 or comprises V185W or comprises V185W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 124, 126, 141, 143, 147, 148, 170, 173, 181, and 183, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0386] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of F118M, S131N, V133A, N138E, Q160F, S162A, D167S, S174N, S176V, T178L, and T180K or comprises an amino acid residue selected from the group consisting of F118M, S131N, V133A, N138E, Q160F, S162A, D167S, S174N, S176V, T178L, and T180K and conservative amino acid substitutions thereof and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124K, F126T, A141T, G143L, K147E, D148K, F170G, V173M, S181M, S183F, and V185W or comprises an amino acid residue selected from the group consisting of S124K, F126T, A141T, G143L, K147E, D148K, F170G, V173M, S181M, S183F, and V185W and conservative amino acid substitutions thereof wherein the amino acid position of each group member is indicated according to the EU numbering.

[0387] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 118, 123, 124, 133, 137, 160, 162, 164, 174, 176, and 178, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 133, 134, 139, 141, 143, 168, 170, 175, 181, 183, and 185, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0388] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 176 or comprises S176Y or comprises S176Y and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 114, 116, 118, 123, 124, 133, 137, 160, 162, 164, 174, and 178, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 183 or comprises S183Y or comprises S183Y and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 124, 126, 133, 134, 139, 141, 143, 168, 170, 175, 181, and 185, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0389] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, S162T, T164V, S174F, S176Y, and T178K or comprises an amino acid residue selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, S162T, T164V, S174F, S176Y, and T178K and conservative amino acid substitutions thereof, and/or the parent domain of the second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, S183Y and V185K or comprises an amino acid residue selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, S183Y, and V185K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0390] In some embodiments the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is a IGKC domain, wherein the amino acid residue which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises V185W and S183V or comprises V185W and S183V and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises S174A or S174A and conservative amino acid substitutions thereof and/or a further amino acid selected from the group consisting of S114E, F116A, F118F, S131T, N137T, S162D, E165L, D167E, T178R, T180R or selected from the group consisting of S114E, P116A, F118F, S131T, N137T, S162D, E165L, D167E, T178R, T180R and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0391] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises S176F and/or T178W or comprises S176F and/or T178W and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises H168F and/or F170A or comprises H168F and/or F170A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0392] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises T178W or comprises T178W and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises L128M or comprises L128M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0393] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IGKC domain and the parent domain of the second engineered immunoglobulin chain is a CH1 domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises T178W or comprises T178W and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises F170A or comprises F170A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0394] In some embodiments the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent, domain of the second engineered immunoglobulin chain is an IGKC domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises S183Y or S183Y and conservative amino acid substitutions thereof and a further amino acid selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, F168K, F170T, Q175D, S181F, and V185K or selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises S174F and/or S176Y or comprises S174F and/or S176Y and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0395] In some embodiments the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is an IGKC domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises S183Y or S183Y and conservative amino acid substitutions thereof and a further amino acid selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K or selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises S162T or S162T and conservative amino acid substitutions thereof and a further amino acid selected from the group consisting of S114Q, F116Y, F181L, E123D, Q124E, V133T, N137K, Q160K, T164V, S174F, S176Y, and T178K or selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, T164V, S174F, S176Y, and T178K and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the EU numbering.

[0396] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 118, 123, 124, 131, 133, 137, 138, 160, 162, 164, 165, 167, 174, 176, 178, and 180, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0397] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114E, S114K, S114Q, P116A, F116T, F116Y, F118M, F118L, E123D, Q124E, S131K, S131T, S131N, V133L, V133A, V133T, N137T, N137E, N137K, N138K, N138E, Q160Y, Q160F, Q160K, S162A, S162G, S162D, S162T, T164V, T164M, E165L, D167E, D167S, S174A, S174M, S174N, S174F, S176V, S176F, S176Y, T178W, T178R, T178L, T178K, T180N, T180R, and T180K or comprises an amino acid residue selected from the group consisting of S114E, S114K, S114Q, F116A, F116T, F116Y, F118M, F118L, E123D, Q124E, S131K, S131T, S131N, V133L, V133A, V133T, N137T, N137E, N137K, N138K, N138E, Q160Y, Q160F, Q160K, S162A, S162G, S162D, S162T, T164V, T164M, E165L, D167E, D167S, S174A, S174M, S174N, S174F, S176V, S176F, S176Y, T178W, T178R, T178L, T178K, T180N, T180R, and T180K, and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0398] In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 128, 133, 134, 139, 141, 143, 147, 148, 168, 170, 173, 175, 181, 183, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0399] In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124E, S124K, S124Q, F126A, F126, F126Y, L128F, L128M, K133D, S134E, T139Q, A141K, A141T, A141N, A141S, G143V, G143A, G143L, G143T, K147T, K147E, D148E, 1D48K, H168Y, H168F, H168K, F170D, F170A, F170G, F170T, V173L, V173M, Q175E, Q175S, Q175D, S181A, S181N, S181M, S181F, S183V, S183F, S183Y, V185W, V185R, V185L, V185K, T187N, T187K and T187R or comprises an amino acid residue selected from the group consisting of S124E, S124K, S124Q, F126A, F126T, F126Y, L128F, L128M, K133D, S134E, T139Q, A141K, A141T, A141N, A141S, G143V, G143A, G143L, G143T, K147T, K147E, D148E, D148K, H168Y, H168F, H168K, F170D, F170A, F170G, F170T, V173L, V173M, Q175E, Q175S, Q175D, S181A, S181N, S181M, S181F, S183V, S183F, S183Y, V185W, V185R, V185L, V185K, T187N, T187K and T187R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0400] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 131, 137, 160, 176, 178 and 180, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 128, 141, 143, 147, 170, 173, 175, 181, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0401] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at position 178 or comprises T178W or comprises T178W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 131, 137, 160, 176, 180, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at position 181 or comprises S181A or comprises S181A and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 124, 126, 128, 141, 143, 147, 170, 173, 175, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0402] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S131K, N137T, Q160Y, S176V, T178W, T180N or comprises an amino acid residue selected from the group consisting of S131K, N137T, Q160Y, S176V, T178W, T180N and conservative amino acid substitutions thereof, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124E, F126A, L128F, A411T, G143V, K147T, F170D, V173L, Q175E, S181A, V185R and T187R or are selected from the group consisting of S124E, F126A, L128F, A141T, G143V, K147T, F170D, V173L, Q175E, S181A, V185R, and T187R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0403] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 131, 137, 162, 165, 167, 174, 176, 178, 180, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 141, 143, 147, 168, 183, 185 and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0404] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises the amino acid residue at position 174 or comprises 174A or comprises 174A and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 114, 116, 131, 137, 162, 165, 167, 178, and 180, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at position 185 or comprises V185W or comprises V185W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 141, 143, 147, 168, 183, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0405] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114E, F116A, S131T, N137T, S162D, E165L, D167E, S174A, T178R, T180R or comprises an amino acid residue selected from the group consisting of S114E, F116A, S131T, N137T, S162, E165L, D167h, S174A, T178R, T180R and conservative amino acid substitutions thereof, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of A141K, G143V, K147T, H168Y, S183V, V185W, and T187N or comprises an amino acid residue selected from the group consisting of A141K, G143V, K147T, H168Y, S183V, V185W, and T187N and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0406] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 131, 133, 137, 138, 162, 164, 174, 176, and 178, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 128, 141, 143, 148, 168, 170, 175, 181, 183, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0407] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises the amino acid residue at position 178 or comprises T178W or comprises T178W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 114, 116, 131, 133, 137, 138, 162, 164, 174, and 176, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at position 183 or comprises S183V or comprises S183V and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 128, 141, 143, 148, 168, 170, 175, 181, 185, and 187, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0408] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114K, F116T, S131T, V133L, N137E, N138K, S162G, T164M, S174M, S176F, and T178W or comprises an amino acid residue selected from the group consisting of S114K, F116T, S131T, V133L, N137E, N138K, S162G, T164M, S174M, S176F, and T178W and conservative amino acid substitutions thereof, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of L128M, A141N, G143A, D148E, H168F, F170A, Q175S, S181N, S183V, V185L, and T187K or comprises an amino acid residue selected from the group consisting of L128M, A141N, G143A, D148E, H168F, F170A, Q175S, S181N, S183V, V185L and T187K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0409] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 118, 131, 133, 138, 160, 162, 167, 174, 176, 178, and 180 and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 141, 143, 147, 148, 170, 173, 181, 183 and 185, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0410] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises the amino acid residue at position 176 or comprises S176V or comprises S176V and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 118, 131, 133, 138, 160, 162, 167, 174, 178, 180, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at position 185 or comprises V185W or comprises V185W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 124, 126, 141, 143, 147, 148, 170, 173, 181, and 183, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0411] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of F118M, S131N, V133A, N138E, Q160F, S162A, D167S, S174N, S176V, and T178L or comprises an amino acid residue selected from the group consisting of FI 18M, S131N, V133A, N138E, Q160F, S162A, D167S, S174N, S176V, and T178L, and conservative amino acid substitutions thereof, and T180K and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124K, F126T, A141T, G143L, K147E, D148K, F170G, V173M, S181M, S183F, and V185W or comprises an amino acid residue selected from the group consisting of S124K, F126T, A141T, G143L, K147E, D148K, F170G, V173M, S181M, S183F, and V185W and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0412] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 114, 116, 118, 123, 124, 133, 137, 160, 162, 164, 174, 176, and 178, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue at a position selected from the group consisting of 124, 126, 133, 134, 139, 141, 143, 168, 170, 175, 181, 183, and 185, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0413] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises the amino acid residue at position 176 or comprises S176Y or comprises S176Y and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 114, 116, 118, 123, 124, 133, 137, 160, 162, 164, 174, and 178, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises the amino acid residue at position 183 or comprises S183Y or comprises S183Y and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 124, 126, 133, 134, 139, 141, 143, 168, 170, 175, 181, and 185, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0414] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, S162T, T164V, S174F, S176Y, and T178K or are selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, S162T, T164V, S174F, S176Y, and T178K and conservative amino acid substitutions thereof, and/or the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises an amino acid residue selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, S183Y, and V185K or are selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, S183Y, and V185K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the EU numbering.

[0415] In some embodiments the parent domain of the further engineered domain of the first and/or second engineered Immunoglobulin chain is a CH1 domain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a IGKC domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises V185W and S183V or comprises V185W and S183V and conservative amino acid substitutions thereof, and/or the amino acid residues which are substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises S174A or S174A and conservative amino acid substitutions thereof and/or a further amino acid selected from the group consisting of S114E, F116A, S131T, N137T, S162D, E165L, D167E, T178R, T180R or are selected from the group consisting of S114E, F116A, S131T, N137T, S162D, E165L, D167E, T178R, T180R and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0416] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises S176F and/or T178W or comprises S176F and/or T178W and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises H168F and/or F170A or comprises H168F and/or F170A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0417] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises T178W or comprises T178W and conservative amino acid substitutions thereof, and/or wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises L128M or comprises L128M and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0418] In some embodiments the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain and the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises T178W or comprises T178W and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises F170A or comprises F170A and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0419] In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises S183Y or comprises S183Y and conservative amino acid substitutions thereof and a further amino acid selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K or are selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises S174F and/or S176Y or comprises S174F and/or S176Y and conservative amino acid substitutions thereof wherein the amino acid position is indicated according to the EU numbering.

[0420] In some embodiments the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is a CH1 domain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is an IGKC domain, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain comprises S183Y or comprises S183Y and conservative amino acid substitutions thereof and a further amino acid selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K or are selected from the group consisting of S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, and V185K and conservative amino acid substitutions thereof, and/or wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the engineered domain of the third engineered immunoglobulin chain comprises S162T or comprises S162T and conservative amino acid substitutions thereof and a further amino acid selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, T164V, S174F, S176Y, and T178K or are selected from the group consisting of S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, T164V, S174F, S176Y, and T178K and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the EU numbering.

[0421] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 842, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 86, 88, and 90,

wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0422] Positions 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 in the protein-protein interface of the CH3 domain as parent domain indicated according to IMGT.RTM. numbering correspond in the same order to positions 347, 349, 351, 364, 366, 370, 371, 392, 394, 397, 399, 405, 407, 409 and 411 indicated according to the EU numbering.

[0423] In some embodiments the parent domain of the first and the second engineered immunoglobulin chain is a CH3 domain.

[0424] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain are CH3 domains of different isotypes.

[0425] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IgG1 CH3 domain, preferably a human CH3 domain from IGHG1 and the parent domain of the second engineered immunoglobulin chain is an IgG3 CH3 domain, preferably a human CH3 domain from IGHG3 or wherein the parent domain of the first engineered immunoglobulin chain is an IgG3 CH3 domain, preferably a human CH3 domain from IGHG3 and the parent domain of the second engineered immunoglobulin chain is an IgG1 CH3 domain preferably a human CH3 domain from IGHG1.

[0426] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and 86, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0427] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is not a charged amino acid, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0428] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is not 88I, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0429] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 85.1 and/or 86 is not a charged amino acid, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0430] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 85.1 and/or 86 are not a charged pair.

[0431] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is 88W or 88W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering

[0432] In some embodiments the CH3 domain is a domain selected from the group consisting of human IgG1 (IGHG1), human IgG2 (IGHG2), human IgG3 (IGHG3) and human IgGP (IGHGP) and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is K88W or K88W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0433] In some embodiments the CH3 domain is a human IgA1 (IGHA1) or a IgA2 (IGHA2) CH3 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 188W or 188W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0434] In some embodiments the CH3 domain is a human IgD (IGHD) CH3 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is V88W or V88W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0435] In some embodiments the CH3 domain is a human IgE CH3 (IGHE) domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is T88W or T88W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0436] In some embodiments the CH3 domain is a human IgG4 (IGHG4) CH3 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is R88W or R88W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0437] In some embodiments the CH3 domain is a human IgM (IGHM) CH3 domain and the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is E88W or E88W and conservative amino acid substitutions thereof and optionally a further amino acid residue selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0438] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88Y, 88K and 88W and/or the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20E, 20S, 20K, 20W, 22A, 22G, 22I, 22L, 22I, 22V, 26R, 26Q, 26T, 26K, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1F, 85.1C, 85.1N, 85.1W, 86S, 86I, 86T, 86H, 86Q, 86V, 86W, 86Y and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0439] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88Y, and 88W, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0440] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20E, 20S, 22A, 22G, 22T, 22L, 22I, 22V, 26Q, 26T, 26K, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1F, 85.1C, 85.1N, 86S, 86I, 86T, 86H, 86Q, 86V, and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0441] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20E, 20S, 20K, 22A, 22G, 22T, 22L, 22I, 22V, 26Q, 26T, 26K, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1F, 85.1C, 85.1N, 86S, 86I, 86T, 86H, 86Q, 86V, and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0442] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88E, 88T, 88Y, and 88W, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0443] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20A, 20N, 20Q, 20E, 20S, 22A, 22G, 22T, 22L, 22I, 22V, 26Q, 26T, 26K, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1S, 85.1R, 85.1H, 85.1K, 85.1F, 85.1C, 85.1N, 86S, 86I, 86H, 86Q, 86V, and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0444] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 5A, 5T, 7F, 7M, 20K, 20N, 20T, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 84L, 84M, 84.2E, 84.2S, 85.1A, 85.1C, 85.1M, 85.1N, 85.1S, 86F, 86S, 86V, 90K, 90N, and 90R or is selected from the group consisting of 3E, 3K, 5A, 5T, 7F, 7M, 20K, 20N, 20T, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 84L, 84M, 84.2E, 84.2S, 85.1A, 85.1C, 85.1M, 85.1N, 85.1S, 86F, 86S, 86V, 90K, 90N, and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0445] In some embodiments relating to a CH3 domain as parent domain of the first and/or the second engineered immunoglobulin chain the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 88I and conservative amino acid substitutions thereof and the optional further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 81W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0446] In some embodiments relating to a CH3 domain as parent domain of the first and/or the second engineered immunoglobulin chain the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 88I and conservative amino acid substitutions thereof and the optional further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 81W and conservative amino acid substitutions and/or the optional further amino acid residue or an additional further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is 86T and conservative amino acid substitutions thereof and/or 84R and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0447] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residues at position 88 and at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0448] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and/or 86 and at position 26 and optionally an amino acid residue at a further position, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0449] In some embodiments the amino acid residue which is substituted at position 85.1 and/or 86 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 85.1A, 85.1S, 85.1C and 85.1N or is selected from the group consisting of 85.1A, 85.1S, 85.1C and 85.1N and conservative amino acid substitutions thereof and/or is selected from the group consisting of 86S and 86V or is selected from the group consisting of 86S and 86V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0450] In some embodiments the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0451] In some embodiments the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 20, 22, 26, 79, 85.1, 86, and 90, and/or the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 84 or is 84L or is 84L and conservative amino acid substitutions thereof, and optionally a further amino acid at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0452] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20K, 22V, 26T, 79Y, 85.1S, 86V, and 90N or is selected from the group consisting of 20K, 22V, 26T, 79Y, 85.1S, 86V, and 90N and conservative amino acid substitutions thereof and/or the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and 86 or comprises 85.1C and conservative amino acid substitutions thereof and 86S and conservative amino acid substitutions thereof, and the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 88, and 90, preferably from the group consisting of 3E, 5A, 7F, 20T, 22V, 26T, 81D, 84L, 84.2E, 88R and 90R or from the group consisting of 3E, 5A, 7F, 20T, 22V, 26T, 81D1, 84L, 84.2E, 88R and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0453] In some embodiments the amino acid residue substitution 85.1C is replaced by amino acid residue substitution 85.1A or 85.1S wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0454] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 3, 5, 20, 22, 26, 27, 81, 84, 85.1, and 86, and/or the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0455] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 84M, 85.1M, 86F or is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 84M, 85.1M, 86F and conservative amino acid substitutions thereof, and/or the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 79 or is 79F or is 79F and conservative amino acid substitutions thereof, and optionally a further amino acid residue at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 81, 842, 85.1, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0456] In some embodiments the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 84M, 85.1M, 86F or is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 84M, 85.1M, 86F and conservative amino acid substitutions thereof, and/or the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and 86 or comprises 85.1N and conservative amino acid substitutions thereof and 86V and conservative amino acid substitutions thereof, and the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 7M, 20N, 22A, 27E, 79F, 81A, 84.25, 88L, and 90K or is selected from the group consisting of 7M, 20N, 22A, 27E, 79F, 81A, 84.2S, 85.1N, 86V, 88L, and 90K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0457] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residue at position 26 and at a further position selected from the group consisting of 3, 22, 27, 79, 81, 84, 85.1, 86, and 88, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0458] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at positions 26 and 86 and optionally at a further position selected from the group consisting of 3, 5, 22, 27, 79, 81, 84, 85.1, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0459] In some embodiments the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprise the amino acid residues at positions 20 and 22, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residues at positions 26 and at a further position selected from the group consisting of 3, 5, 22, 27, 79, 81, 84, 85.1, 86, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0460] In some embodiments the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprise the amino acid residues at positions 20 and 22, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residues at positions 26 and 86 and optionally at a further position selected from the group consisting of 3, 5, 22, 27, 79, 81, 84, 85.1, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0461] In some embodiments the amino acid residue which is substituted at position 20 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20N and 20T, and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0462] In some embodiments the amino acid residue which is substituted at position 26 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 26T and 26E and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0463] In some embodiments the amino acid residue which is substituted at position 22 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A and 22V, and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0464] In some embodiments the amino acid residue which is substituted at position 86 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 86V and 86F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0465] In some embodiments the amino acid residue which is substituted at position 20 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20K, 20S, 20W and 20E and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22L, 22I, 22V, 22T, 26K, 26R, 26Q, 26T, 26V, 26S, 26N, 26E, 85.1W, 85.1F, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1C, 85.1N, 86W, 86Y, 86S, 86I, 86H, 86Q, 86V, 86T, 86F, 88Q, 88L, 88V, 88R, 88E, 88T, 88I, 88Y, 88K and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0466] In some embodiments the amino acid residue which is substituted at position 20 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, and 20E and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22L, 22I, 22V, 26Q, 26T, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1M, 85.1K, 85.1C, 85.1N, 86S, 86I, 86T, 86H, 86Q, 86V, 86T, 86F, 88Q, 88L, 88V, 88R, 88E, 88T, 88I, 88Y, and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0467] In some embodiments the amino acid residue which is substituted at position 20 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20K, 20S and 20E and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22L, 22I, 22V, 26K, 26Q, 26T, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1C, 85.1N, 86S, 86I, 86H, 86Q, 86V, 86T, 86F, 88Q, 88L, 88V, 88R, 88E, 88T, 88I, 88Y, and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0468] In some embodiments the amino acid residues which are substituted at position 20 and position 22 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain are selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20K, 20S, 20W and 20E and 22A, 22G, 22L, 22I, 22V, 22T and/or wherein the further amino acid residue substituted in the protein-protein Interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 26K, 26R, 26Q, 26T, 26V, 26S, 26N, 26E, 85.1W, 85.1F, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1I, 85.1K, 85.1C, 85.1N, 86W, 86Y, 86S, 86I, 86H, 86Q, 86V, 86T, 86F, 88Q, 88L, 88V, 88R, 88E, 88T, 88I, 88Y, 88K and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0469] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 5A, 5T, 7F, 7M, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 84L, 84M, 84.2E, 84.2S, 85.1A, 85.1C, 85.1M, 85.1N, 85.1S, 86F, 86S, 86V, 88L, 88R, 88W, 90K, 90N, and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0470] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 5A, 5T, 7F, 7M, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 84L, 84M, 84.2E, 84.2S, 85.1A, 85.1C, 85.1M, 85.1N, 85.1S, 86F, 86S, 86V, 88L, 88R, 88W, 90K, 90N, and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0471] In some embodiments the amino acid residue which is substituted at position 26 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 26K, 26R, 26Q, 26T, 26V, 26S, 26N, 26E and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22L, 22I, 22V, 22T, 85.1W, 85.1F, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1C, 85.1N, 86W, 86Y, 86S, 86I, 86H, 86Q, 86V, 86T, 86F, 88Q, 88L, 88V, 88R, 88E, 88T, 88I, 88Y, 88K and 88W wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0472] In some embodiments the amino acid residues which are substituted at position 26 and position 86 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 26K, 26R, 26Q, 26T, 26V, 26S, 26N, 26E and 86W, 86Y, 86S, 86I, 86H, 86Q, 86V, 86T, 86F and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22L, 22I, 22V, 22T, 85.1W, 85.1F, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1C, 85.1N, 88Q, 88L, 88V, 88R, 88E, 88T, 88I, 88Y, 88K and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0473] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 22A, 22L, 22V, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 84L, 84M, 85.1A, 85.1C, 85.1M, 85.1N, 85.1S, 86F, 86S, 86V, 88L, 88R, and 88W, and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0474] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86 and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3 and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1 and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 86 and 90, wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering. Positions 3, 5, 7, 20, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86 and 90 in the protein-protein interface of the CH2 domain as parent domain indicated according to IMGT.RTM. numbering correspond in the same order to positions 239, 241, 243, 258, 260, 264, 265, 288, 290, 292, 293, 295, 296, 301, 303, 305, 307 indicated according to the EU numbering.

[0475] In some embodiments the parent domain of the first and the second engineered immunoglobulin chain is a CH2 domain.

[0476] In some embodiments the parent domains of the first and the second engineered immunoglobulin chain are CH2 domains of different isotypes.

[0477] In some embodiments the parent domain of the first engineered immunoglobulin chain is an IgG1 CH2 domain, preferably a human CH2 domain from IGHG1 and the parent domain of the second engineered immunoglobulin chain is an IgG3 CH2 domain, preferably a human CH2 domain from IGHG3 or wherein the parent domain of the first engineered immunoglobulin chain is an IgG3 CH2 domain, preferably a human CH2 domain from IGHG3 and the parent domain of the second engineered immunoglobulin chain is an IgG1 CH2 domain preferably a human CH2 domain from IGHG1.

[0478] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is 88W or 88W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0479] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain at position 85.1 and/or 86 is selected from the group consisting of 85.1A and 85.1N or is selected from the group consisting of 85.1A and 85.1N and conservative amino acid substitutions thereof, and/or is 86S or 86S and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0480] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88I, 88T, 88K, 88E, 88Y, and 88W and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20K, 20W, 20S, 20E, 20Q, 22A, 22G, 22T, 22L, 22V, 26Q, 26T, 26K, 26V, 26S, 26R, 26N, 26E, 85.1R, 85.1H, 85.1K, 85.1T, 85.1M, 85.1A, 85.1S, 85.1W, 85.1F, 85.1C, 85.1N, 86S, 86I, 86H, 86T, 86W, 86Y, 86V, 86Q and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0481] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88I, 88T, 88K, 88E, 88Y, and 88W and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20K, 20W, 20S, 22A, 22C, 22T, 22L, 22V, 26Q, 26T, 26K, 26V, 26S, 26R, 26N, 26E, 85.11T, 85.1M, 85.1A, 85.1S, 85.1W, 85.1F, 85.1C, 85.1N, 86S, 86I, 86H, 86T, 86W, 86Y, 86V, and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0482] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 5A, 5T, 5Y, 7M, 7L, 20K, 20N, 20T, 20S, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 83M, 83T, 84L, 84.2E, 84.2S, 84.3D, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86F, 86Y, 90K, 90N, and 90R or is selected from the group consisting of 3E, 3K, 5A, 5T, 5Y, 7M, 7L, 20K, 20N, 20T, 20S, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 83M, 83T, 84L, 84.2E, 84.2S, 84.3D, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86F, 86Y, 90K, 90N, and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0483] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residues at position 88 and at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86 and 90, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0484] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and/or 86 and at position 26 and optionally an amino acid residue at a further position, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0485] In some embodiments the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 20, 22, 26, 79, 85.1, and 90, and/or the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 84 or is 84L or is 84L and conservative amino acid substitutions thereof, and optionally a further amino acid at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0486] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20K, 22V, 26T, 79Y, 85.1S, and 90N or is selected from the group consisting of 20K, 22V, 26T, 79Y, 85.1S, and 90N and conservative amino acid substitutions thereof and/or the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain at position 85.1 or is 85.1C or is 85.1C and conservative amino acid substitutions thereof, and wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain at position 86 or is 86S or is 86S and conservative amino acid substitutions thereof and wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 3, 5, 20, 22, 26, 81, 84, 84.2, 88, and 90, preferably from the group consisting of 3E, 5A, 20T, 22V, 26T, 81D, 84L, 84.2E, 88R and 90R or from the group consisting of 3E, 5A, 20T, 22V, 26T, 81D, 84L, 84.2E, 88R and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0487] In some embodiments the amino acid residue substitution 85.1C is replaced by amino acid residue substitution 85.1A or 85.1 wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0488] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 3, 5, 20, 22, 26, 27, 81, 83, 85.1, and 86, and/or the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0489] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 83M, 85.1M, and 86F or is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 83M, 85.1M, and 86F and conservative amino acid substitutions thereof, and/or the further amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 79 or is 79F or is 79F and conservative amino acid substitutions thereof and optionally a further amino acid residue at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 85.1, 88, and 90, or at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0490] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 83M, 85.1M, and 86F or is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 83M, 85.1M, and 86F and conservative amino acid substitutions thereof, and/or the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain at position 85.1 is 85.1N or is 85.1N and conservative amino acid substitutions thereof, and wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain at position 22 is 22A or is 22A and conservative amino acid substitutions thereof, and/or wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 7M, 20N, 27E, 79F, 81A, 84.2S, 88L, and 90K or is selected from the group consisting of 7M, 20N, 27E, 79F, 81A, 84.2S, 88L, and 90K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0491] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 5, 7, 20, 83, 84.3, 85.1, and 86 and/or the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at a position selected from the group consisting of 5, 7, 20, 83, 84.3, and 88 or at a position selected from the group consisting of 5, 7, 20, 83, 84.3, 85.1, and 88, or at a position selected from the group consisting of 5, 7, 20, 83, 84.3, 86, and 88 wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0492] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 5Y, 7L, 20S, 83T, 84.3D, 85.1F, and 86Y or is selected from the group consisting of 5Y, 7L, 20S, 83T, 84.3D, 85.1F, and 86Y and conservative amino acid substitutions thereof, and/or the further amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 20 or is 20S or is 20S and conservative amino acid substitutions thereof, and optionally a further amino acid residue at a position selected from the group consisting of 5, 7, 83, 84.3, and 88 or at a position selected from the group consisting of 5.7, 83, 84.3, 85.1 and 88 or at a position selected from the group consisting of 5, 7, 83, 84.3, 86 and 88, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0493] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 5Y, 7L, 20S, 83T, 84.3D, 85.1F and 86Y or is selected from the group consisting of 5Y, 7L, 20S, 83T, 84.3D, 85.1F and 86Y and conservative amino acid substitutions thereof, and/or the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 85.1 or is 85.1F or is 85.1F and conservative amino acid substitutions thereof, and wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 20 or is 20S or is 20S and conservative amino acid substitutions thereof, and/or wherein a further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 5Y, 7L, 83T, 84.3D, 86Y, and 88K or is selected from the group consisting of 5Y, 7L, 83T, 84.3D, 86Y and 88K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0494] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86, 88 and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 26 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 27, 79, 81, 83, 84, 84.2, 84.3, 85.1, 86, 88 and 90, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0495] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH2 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, wherein the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain are at position 26 and at a further position selected from the group consisting of 3, 22, 27, 79, 81, 84, 85.1, 86, and 88, wherein the amino acid residue substituted at position 20 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 26 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0496] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at positions 26 and 86 and optionally at a further position selected from the group consisting of 3, 5, 22, 27, 79, 81, 84, 85.1, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0497] In some embodiments the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprise the amino acid residues at positions 20 and 22, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residues at positions 26 and at a further position selected from the group consisting of 3, 5, 22, 27, 79, 81, 84, 85.1, 86, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0498] In some embodiments the amino acid residues which are substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprise the amino acid residues at positions 20 and 22, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, the amino acid residues which are substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprise the amino acid residues at positions 26 and 86 and optionally at a further position selected from the group consisting of 3, 5, 22, 27, 79, 81, 84, 85.1, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0499] In some embodiments the amino acid residue which is substituted at position 20 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20N and 20T, and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering. In some embodiments the amino acid residue which is substituted at position 26 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 26T and 26E and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0500] In some embodiments the amino acid residue which is substituted at position 22 in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A and 22V, and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0501] In some embodiments the amino acid residue which is substituted at position 86 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 86V and 86F and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0502] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 20 is selected from the group consisting of 20V, 20T, 20A, 20N, 20K, 20Q, 20E, 20W and 20S and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22T, 22L, 22V, 22I, 26Q, 26T, 26K, 26V, 26S, 26R, 26N, 26E, 85.1R, 85.1H, 85.1K, 85.1T, 85.1M, 85.1A, 85.1S, 85.1W, 85.1F, 85.1C, 85.1N, 86Q, 86S, 86I, 86H, 86T, 86W, 86Y, 86V, 86F, 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88K, 88Y, and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0503] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 20 is selected from the group consisting of 20V, 20T, 20A, 20N, 20K, 20W and 20S and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 22A, 22G, 22T, 22L, 22V, 26Q, 26T, 26K, 26V, 26S, 26R, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1W, 85.1F, 85.1C, 85.1N, 86S, 86I, 86H, 86T, 86W, 86Y, 86V, 86F, 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88K, 88Y, and 88W, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0504] In some embodiments the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 5A, 5T, 5Y, 7M, 7L, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 83M, 83T, 84L, 84.2E, 84.2S, 84.3D, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86F, 86Y, 88K, 88L, 88R, 88W, 90K, 90N, and 90R or selected from the group consisting of 3E, 3K, 5A, 5T, 5Y, 7M, 7L, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81I, 83M, 83T, 84L, 84.2E, 84.2S, 84.3D, 85.1S, 85.1A, 85.1N, 85.1M, 85.1F, 86S, 86F, 86Y, 88K, 88L, 88R, 88W, 90K, 90N, and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0505] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH4 domain, wherein the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 20, 22, 85.1, 86, and 88, and/or wherein the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises an amino acid residue substitution at a position selected from the group consisting of 22, 26, 85.1, 86, and 88, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering. In some embodiments the parent domain of the first and the second engineered immunoglobulin chain is a naturally occurring CH4 domain.

[0506] In some embodiments the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is 88W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

[0507] In some embodiments preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues, more particular at least six, even more particular at least seven, most particular all amino acid residues of the protein-protein interface of the parent domain of the first and/or the second engineered immunoglobulin chain are substituted. Equally preferred two, more preferably three, most preferably four, in particular five amino acid residues, more particular six, even more particular seven amino acid residues of the protein-protein interface of the parent domain of the first and/or the second engineered immunoglobulin chain are substituted. Equally preferred less than two, more preferably less than three, most preferably less than four, in particular less than five amino acid residues, more particular less than six, even more particular less than seven amino acid residues of the protein-protein interface of the parent domain of the first and/or the second engineered immunoglobulin chain are substituted.

[0508] In some embodiments at least one, preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues, more particular at least six, even more particular at least seven, most particular all amino acid residues of the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain

[0509] In some embodiments the amino acid residues of the protein-protein interface of the parent domain of the first and/or second engineered immunoglobulin chain which are substituted are not adjacent.

[0510] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein, at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues of the protein-protein interface of the parent domain of the first and/or second engineered immunoglobulin chain which are substituted are not adjacent.

[0511] In some embodiments the amino acid sequence of the engineered domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain do not contain an insertion of one or more amino acid residues compared to the amino acid sequence of the parent domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain.

[0512] In some embodiments the amino acid sequence of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain do not contain an insertion of one or more amino acid residues compared to the amino acid sequence of the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin.

[0513] In some embodiments the amino acid sequence of the engineered domain of the third engineered immunoglobulin chain do not contain an insertion of one or more amino acid residues compared to the amino acid sequence of the parent domain of the third engineered immunoglobulin chain.

[0514] In some embodiments the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain comprise a further engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said further engineered domain of said first engineered immunoglobulin chain and/or said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a third member of the naturally occurring immunoglobulin super-family, and

wherein the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is interacting with the protein-protein interface of an engineered domain of a third engineered immunoglobulin chain by hetero-dimerization or by homo-dimerization, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said engineered domain of said third engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a fourth member of the naturally occurring immunoglobulin super-family, and wherein the donor domain of the third member of the naturally occurring immunoglobulin super-family and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and of the engineered domain of the third engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain and of the engineered domain of the second engineered immunoglobulin chain.

[0515] Preferably, the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain are not identical.

[0516] Preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues, more particular at least six, even more particular at least seven, most particular all amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain and/or the parent domain of the engineered domain of the third engineered immunoglobulin chain are substituted. Equally preferred two, more preferably three, most preferably four, in particular five amino acid residues, more particular six, even more particular seven amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain and/or the parent domain of the engineered domain of the third engineered immunoglobulin chain are substituted. Equally preferred less than two, more preferably less than three, most preferably less than four, in particular less than five amino acid residues, more particular less than six, even more particular less than seven amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain and/or the parent domain of the engineered domain of the third engineered immunoglobulin chain are substituted.

[0517] In some embodiments at least one, preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues, more particular at least six, even more particular at least seven, most particular all amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first and/or second engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the third engineered immunoglobulin chain.

[0518] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain which are substituted are not adjacent. In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein, at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first engineered immunoglobulin chain and the parent domain of the engineered domain of the third engineered immunoglobulin chain which are substituted are not adjacent.

[0519] In some embodiments the third engineered immunoglobulin chain comprises a further engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said further engineered domain of said third engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a fifth member of the naturally occurring immunoglobulin super-family, and

wherein the protein-protein interface of the further engineered domain of the third engineered immunoglobulin chain is interacting with the protein-protein interface of an engineered domain of a fourth engineered immunoglobulin chain by hetero-dimerization or by homo-dimerization, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said engineered domain of said third engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a sixth member of the naturally occurring immunoglobulin super-family, and wherein the donor domain of the fifth member of the naturally occurring immunoglobulin super-family and the donor domain of the sixth member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the protein-protein interface of the further engineered domain of the third engineered immunoglobulin chain and of the engineered domain of the fourth engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain and of the engineered domain of the second engineered immunoglobulin chain and from the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and from the protein-protein interface of the engineered domain of a third engineered immunoglobulin chain.

[0520] The parent domains of the further engineered domain of the third engineered immunoglobulin chain and/or of the engineered domain of the fourth engineered immunoglobulin chain is selected from consisting of CH1, CH2, CH3, CH4, IGKC, IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7, wherein the amino acid residues of the protein-protein interface of the further engineered domain of the third engineered immunoglobulin chain and/or of the engineered domain of the fourth engineered immunoglobulin chain which are substituted are e.g. selected from the group of the amino acid residues of the protein-protein interface for the parent domains of the first and/or second engineered immunoglobulin chains as described supra.

[0521] The donor domain of the fifth and/or a sixth member of the naturally occurring immunoglobulin super-family are e.g. selected from for the group of donor domain of the first and/or second member of the naturally occurring immunoglobulin super-family as described supra.

[0522] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 13 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, preferably SEQ ID NO: 14 or SEQ ID NO: 15.

[0523] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 13 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 14.

[0524] Thus in a farther aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ 11D NO: 13 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface.

[0525] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 13, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 13 contains at least two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of K24, V26, T30, Y52, S63, V67, W69, and N71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 13 in the sequence listing, in a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 14, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 14 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of E7, A9, F11, T24, V26, T30, D54, L57, E59, A65, S67, R69, and R71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 14 in the sequence listing.

[0526] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 15.

[0527] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 15, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 15 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of E7, A9, F11, T24, V26, T30, D54, L57, E59, C65, S67, R69, and R71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 15 in the sequence listing.

[0528] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 16, wherein the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by hetero-dimerization.

[0529] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 16, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 16 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of E7, A9, F11, T24, V26, T30, D54, L57, E59, S65, S67, R69, and R71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 16 in the sequence listing.

[0530] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein the first engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 3 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by hetero-dimerization.

[0531] In some embodiments the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 3, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 3 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of S144K, T146V, K150T, K172Y, F185S, Y187V, K189W, and T191N, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 3 in the sequence listing.

[0532] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein the second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 4, wherein the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by hetero-dimerization.

[0533] In some embodiments the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 4, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 4 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven or eight or nine or ten, or eleven or twelve, most particular all of the amino acid residues selected from the group consisting of Q127E, Y129A, L131F, S144T, T146V, K150T, T174D, V177L, D179E, F185C, Y187S, K189R, and T191R, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 4 in the sequence listing.

[0534] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 34.

[0535] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 35 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface.

[0536] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 35, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 35 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of K127, T129, T144, L146, E150, K151, G174, M177, M185, F187, and W189, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 35 in the sequence listing.

[0537] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 34.

[0538] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 34, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 34 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of M131, N144, A146, E151, F172, A174, S179, N185, V187, L189, and K191, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 34 in the sequence listing.

[0539] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO:40 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 39.

[0540] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 40 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface.

[0541] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 40, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 40 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting K7, T9, T24, L26, E30, K31, G55, M57, M67, F69, and W71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 40 in the sequence listing.

[0542] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO 39.

[0543] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 39, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 39 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of M11, N24, A26, E31, F51, A53, S58, N64, V66, L68, and K70, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 39 in the sequence listing.

[0544] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 48 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 49.

[0545] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 48 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface.

[0546] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 48, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 48 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of Q7, Y9, D16, E17, Q22, S24, T26, K51, T53, D58, F64, Y66, and K68, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 48 in the sequence listing.

[0547] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 49.

[0548] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 49, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 49 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of Q7, Y9, L11, D16, E17, T26, K30, K53, T55, V57, F67, Y69, and K71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 49 in the sequence listing.

[0549] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO; 76 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 14.

[0550] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 76, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 76 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of K24, V26, T30, Y52, 565, V67, W69, and N71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 76 in the sequence listing.

[0551] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 14, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 14 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of E7, A9, F11, T24, V26, T30, D54, L57, E59, A65, S67, R69, and R71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 14 in the sequence listing.

[0552] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 78 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 79. In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 78 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface.

[0553] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 78, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 78 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of E7, H9, R30, R57, E59, A65, and T67, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 78 in the sequence listing.

[0554] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 79.

[0555] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 79, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 79 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of T24, L52, W54, I69, and R71, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 79 in the sequence listing.

[0556] In some embodiments the first engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 84 and/or the second engineered immunoglobulin chain comprises an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 85.

[0557] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 84 and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface.

[0558] In some embodiments the engineered domain with a protein-protein interface of the first engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 84, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 84 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of K26, V28, Y56, S71, V73, W75, and N77, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 84 in the sequence listing.

[0559] In a further aspect the present invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface and a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface comprising the amino acid sequence of SEQ ID NO: 85.

[0560] In some embodiments the engineered domain with a protein-protein interface of the second engineered immunoglobulin chain comprises an amino acid sequence which is at least 80%, preferably at least 90, more preferably at least 95%, most preferred at least 96%, in particular at least 97%, more particular at least 98%, most particular at least 99% identical to the amino acid sequence of SEQ ID NO: 85, with the proviso that the amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 85 contains at least one, usually two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven, most particular all of the amino acid residues selected from the group consisting of E8, A10, F12, V28, D58, L61, A71, S73, R75, and R77, wherein the amino acid position of each group member is indicated according to the numbering of the amino acid residues of SEQ ID NO: 85 in the sequence listing.

[0561] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 7, 13-29, 34, 35, 36, 39, 40, 41, 42, 48, 49, 50, 51, 59, 61, and 68 and/or the second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 7, 13-29, 34, 35, 36, 39, 40, 41, 42, 48, 49, 50, 51, 59, 61, and 68.

[0562] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 7, 13-29, 34, 35, 36, 39, 40, 41, 42, 48, 49, 50, 51, 59, 61, 68, 73, 78 and 84 and/or the second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 7, 13-29, 34, 35, 36, 39, 40, 41, 42, 48, 49, 50, 51, 59, 61, 68, 74, 79 and 85.

[0563] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 13, 59, and 68 and/or the second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 14, 13, 16, 17, 41, 50, and 61.

[0564] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 13, 59, 68, 73 and 84 and/or the second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 14, 15, 16, 17, 41, 50, 61, 74 and 85.

[0565] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 39, and 41 and/or the second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 40, and 42.

[0566] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 49, 50, and 51 and/or the second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 49, 50, and 51. Preferably the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 48 and the second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 49 or of SEQ ID NO: 51. Equally preferred the first engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 50 and the second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 49 or of SEQ ID NO: 51.

[0567] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 78 and/or the second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 79.

[0568] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first and/or second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting o SEQ ID NOs: 3, 7, 13, 59, and 68 and the third engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 14, 15, 16, 17, 41, 50, and 61.

[0569] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first and/or second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 39 and 41 and/or the third engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs 35, 36, 40, and 42.

[0570] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first and/or second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 7, 13-29, 34, 35, 36, 39, 40, 41, 42, 48, 49, 50, 51, 59, 61, 68, 73, 78 and 84 and/or the third engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs 3, 4, 6, 7, 13-29, 34, 35, 36, 39, 40, 41, 42, 48, 49, 50, 51, 59, 61, 68, 74, 79 and 85.

[0571] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first and/or second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 78 and/or the third engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO 79.

[0572] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first and/or second engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 13, 59, 68 and/or the third engineered immunoglobulin chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 13, 59, 68.

[0573] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 72, and the second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 71 and the third engineered immunoglobulin chain which interacts with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 51.

[0574] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof which comprises a first engineered immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 72, a second engineered immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 70, a third engineered immunoglobulin chain with an engineered domain which interacts with the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 69 and a third engineered immunoglobulin chain with an engineered domain which interacts with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 42.

[0575] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and second engineered immunoglobulin chains comprise an Fe region. In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and/or the second and the third engineered immunoglobulin chains comprise an Fc region. Usually the Fc region is selected from the group consisting of IgA, IgE, IgD, IgG, and IgM Fc region.

[0576] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and second engineered immunoglobulin chains comprise an Fc region from the same species, preferably from the same species and isotype, more preferably from the same species, isotype and subclass.

[0577] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and/or the second and the third engineered immunoglobulin chains comprise an Fc region from the same species, preferably from the same species and isotype, more preferably from the same species, isotype and subclass.

[0578] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the Fc regions of the first and second engineered immunoglobulin chains are are not identical. In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the Fc regions of the first and second engineered immunoglobulin chains have amino acid sequences which are different from each other by one amino acid or by two amino acids or by three amino acids or by four amino acids or by five amino acids or by five to ten amino acids or by ten to thirty amino acids, preferably by at least two amino acids or by at least three amino acids or by at least four amino acids or by at least five amino acids or by at least five to ten amino acids or by at least ten to thirty amino acids.

[0579] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof which is bispecific.

[0580] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the hetero-dimeric immunoglobulin or fragment thereof is a full-length antibody.

[0581] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the hetero-dimeric immunoglobulin or fragment thereof is a full-length antibody which is bispecific.

[0582] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody binds to antigens selected from the group consisting of HER2, EGFR, CD19 and VLA-2. Preferably the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody is specific to HER2 and EGFR. Equally preferably the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody is specific to CD19 and VLA-2.

[0583] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof which is a Fab, with a first engineered immunoglobulin chain and a second engineered immunoglobulin chain.

[0584] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof is a F(ab').sub.2 fragment, wherein the first engineered immunoglobulin chain comprises an engineered domain and a further engineered domain, whereas the protein interface of the engineered domain of the first engineered immunoglobulin chain interacts with an engineered domain of a third immunoglobulin chain and the protein interface of the further engineered domain interacts with an engineered domain of a third immunoglobulin chain, wherein the protein interfaces of these engineered domain of these two third immunoglobulin chains are the same or are different form each other.

[0585] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof, wherein wherein at least one additional polypeptide is fused to the first and/or second engineered immunoglobulin chain. Preferably the one additional polypeptide is fused to the first engineered immunoglobulin chain. Suitably the additional polypeptide is selected from the group consisting of Fab, scFv, diabody, domain antibody, pharmacologically active peptide or protein, receptor extracellular domain, CDR grafted polypeptide and therapeutic engineered protein scaffold. Fusion is usually achieved by genetic engineering as known to the skilled person and may involve amino acid sequence linkers which do not form part of the amino acid sequences to be fused.

[0586] In some embodiments the present disclosure provides a multidomain protein comprising at least a first and a second nonidentical engineered domain, each of the first and the second engineered domain containing a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the first parent domain are substituted with amino acid residues at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family and at least one amino acid residue of the protein-protein interface of the second parent domain are substituted with amino acid residues at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family,

wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a hetero-dimer or a homo-dimer.

[0587] The multidomain protein comprises preferably a Fc region. Usually the Fc region is selected from the group consisting of IgA, IgE IgD, IgG, and IgM Fc region.

[0588] In some embodiments the present disclosure provides a hetero-dimeric immunoglobulin or fragment thereof or methods of making hetero-dimeric immunoglobulins or fragments thereof as described below wherein the amino acid residue of the protein-protein interlace of the parent domain of the parent immunoglobulin chain which is substituted at the equivalent 3D structural position from the protein-protein interface of a donor domain is an amino acid residue in the protein-protein interface of a donor domain which when overlaid on the protein-protein interface of the parent domain by superimposing the carbon alpha traces of both domains, occupies a 3D position within less than a distance of 6 .ANG. to the closest residue of the parent domain, wherein the donor domain is different from the parent domain.

Methods of Making Hetero-Dimeric Immunoglobulins or Fragments Thereof

[0589] In a further aspect the present invention provides a method to produce a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising:

(a) substituting at least one amino acid residue of the protein-protein interface of a parent domain of a first parent immunoglobulin chain at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family to obtain a first engineered immunoglobulin chain comprising an engineered domain, (b) substituting at least one amino acid residue of the protein-protein interface of the parent domain of a second parent immunoglobulin chain at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family to obtain a second engineered immunoglobulin chain comprising an engineered domain, (c) culturing a host cell comprising a nucleic acid encoding said engineered immunoglobulin chains, wherein the culturing is such that the nucleic acid is expressed and the engineered immunoglobulin chains produced; and (d) recovering the hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof from the host cell culture.

[0590] In some embodiments the present disclosure provides a method wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical.

[0591] In some embodiments the present disclosure provides a method wherein the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by hetero-dimerization or by homo-dimerization.

[0592] In some embodiments the present disclosure provides a method comprising a parent domain of the first and/or second parent immunoglobulin chain which is selected from the group consisting of CH1, CH2, CH3, CH4, IGKC, IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7, CH1, CH3 can be from IGHA1, IGHA2, IGHD, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP and IGHM, CH4 can be from IGHE and IGHM. CH2 can be from IGHA1, IGHA2, IGHD, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP and IGHM. In some embodiments the parent domain of the first and/or the second engineered immunoglobulin chain is a domain selected from the group consisting of CH1 domain, CH4 domain, IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain, preferably from the group consisting of CH1 domain, IGKC domain, IGLC1 domain, IGLC2 domain, IGLC3 domain, IGLC6 domain, and IGLC7 domain.

[0593] Usually the parent domain of the first and/or second engineered immunoglobulin chain is from human. Preferably the parent domain of the first and/or second engineered immunoglobulin chain is from human and from the same isotype, species and subclass. The preferred parent domain of the first and/or second engineered immunoglobulin chain, in particular the parent domain of the first and second engineered immunoglobulin chain, is a CH3 domain, more preferably a human CH3 domain, in particular a human CH3 domain from IGHG1, Equally preferred the parent domain of the first engineered immunoglobulin chain is a CH1 domain and the parent domain of the second engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain. Equally, the preferred parent domain of the first engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain and the preferred parent domain of the second engineered immunoglobulin chain is a CH1 domain more preferably a human CH1 domain selected from the group consisting of IGHG1, IGHG2, IGHG3, and IGHG4, in particular a human CH1 domain from IGHG1.

[0594] In some embodiments the present disclosure provides a method, wherein the parent domains of the first and the second engineered immunoglobulin chain form a naturally occurring homo-dimer. In some embodiments the present disclosure provides a method, wherein the parent domains of the first and the second engineered immunoglobulin chain form a naturally occurring hetero-dimer.

[0595] In some embodiments the present disclosure provides a method, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and/or wherein the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is interacting with the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain by hetero-dimerization or by homo-dimerization.

[0596] Preferably the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer.

[0597] In some embodiments the present disclosure provides a method, wherein the amino acid residues from the protein-protein interface of the donor domain of the first and second member of the naturally occurring immunoglobulin super-family are amino acids non essential to the core integrity of the domain.

[0598] In some embodiments the present disclosure provides a method, wherein the first and the second member of the naturally occurring immunoglobulin super-family are selected from the TCR constant domain family. Preferably the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2). Equally the donor domain of the first member of the naturally occurring immunoglobulin super-family can be the TCR constant domain beta (SEQ ID NO: 2) and the donor domain of the second member of the naturally occurring immunoglobulin super-family can be the TCR constant domain alpha (SEQ ID NO: 1). In a further embodiment the present disclosure provides a method, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2), wherein the cysteine (C) at amino acid position 75 in SEQ ID NO: 2 is substituted with alanine (A) or serine (S), preferably with alanine (A).

[0599] In a further embodiment the present disclosure provides a method, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) or wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0600] In some embodiments the present disclosure provides a method, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2), the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0601] In some embodiments the present disclosure provides a method, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33), the donor domain of the third member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2).

[0602] In some embodiments the present disclosure provides a method, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) or the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2) or the TCR constant domain gamma (SEQ ID NO: 33), the donor domain of the third and the fourth member of the naturally occurring immunoglobulin super-family is the IgG1 CH3 domain (SEQ ID NO: 47), with the proviso that if the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2) and if the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32), the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33).

[0603] In a further embodiment the present disclosure provides a method, wherein the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain comprise a further engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said further engineered domain of said first engineered immunoglobulin chain and/or said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a third member of the naturally occurring immunoglobulin super-family, and

wherein the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is interacting with the protein-protein interface of an engineered domain of a third engineered immunoglobulin chain by hetero-dimerization or by homo-dimerization, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said engineered domain of said third engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a fourth member of the naturally occurring immunoglobulin super-family and, wherein the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and of the engineered domain of the third engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain and of the engineered domain of the second engineered immunoglobulin chain.

[0604] In some embodiments the present disclosure provides a method, wherein the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and the engineered domain of the third engineered immunoglobulin chain are not identical.

[0605] In some embodiments the parent domain of the first and/or second engineered immunoglobulin chain, in particular the parent domain of the first and second engineered immunoglobulin chain is a CH3 domain, preferably a CH3 domain of the same species, isotype and subclass, and the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin is a CH1 domain and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a a light chain constant domain such as an IGLC domain or an IGKC domain.

[0606] In some embodiments the parent domain of the first engineered immunoglobulin chain is a light chain constant domain such as an IGLC domain or an IGKC domain and the parent domain of the engineered domain of the second engineered immunoglobulin chain is a CH1 domain and the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin and the parent domain of the engineered domain of the third engineered immunoglobulin chain is a CH3 domain, preferably a CH3 domain of the same species, isotype and subclass.

[0607] In some embodiments the present disclosure provides a method, wherein the amino acid residues of the protein-protein interface of the parent domain of the first and/or second parent immunoglobulin chain which are substituted are not adjacent.

[0608] In some embodiments the present disclosure provides a method, wherein the amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin and/or the parent domain of the engineered domain of the third engineered immunoglobulin chain which are substituted are not adjacent.

[0609] In some embodiments the present disclosure provides a method, wherein, preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues of the protein-protein interface of the parent domain of the first and/or second engineered immunoglobulin chain which are substituted are not adjacent.

[0610] In some embodiments the present disclosure provides a method, wherein, preferably at least two, more preferably at least three, most preferably at least four, in particular at least five amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin and/or the parent domain of the engineered domain of the third engineered immunoglobulin chain which are substituted are not adjacent.

[0611] In some embodiments the present disclosure provides a method, wherein preferably at least two, more preferably at least three, most preferably at least four, in particular at least five more particular at least six, most particular at least seven amino acid residues of the protein-protein interface of the parent domain of the first and/or second parent immunoglobulin chain are substituted.

[0612] In some embodiments the present disclosure provides a method, wherein preferably at least two, more preferably at least three, most preferably at least four, in particular at least five more particular at least six, most particular at least seven amino acid residues of the protein-protein interface of the parent domain of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin and/or the parent domain of the engineered domain of the third engineered immunoglobulin chain are substituted.

[0613] In some embodiments the present disclosure provides a method, wherein the amino acid sequence of the engineered domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain do not contain an insertion of one or more amino acid residues compared to the amino acid sequence of the parent domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain.

[0614] In some embodiments the present disclosure provides a method, wherein at least one 3D structural position of the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain is different in amino acid residue compared to the identical 3D structural position of the protein-protein interface of the engineered domain of the second engineered immunoglobulin chain.

[0615] In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains have parent domains from the same species, preferably from the same species and isotype, more preferably from the same species, isotype and subclass. In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains have identical parent domains, i.e. parent domains which have the identical amino acid sequence.

[0616] In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains have parent domains from IgG1, preferably human IgG1.

[0617] In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains have parent domains which are not identical. In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains have parent domains which have amino acid sequences which are different from each other by one amino acid or by two amino acids or by three amino acids or by four amino acids or by five amino acids or by five to ten amino acids or by ten to thirty amino acids

[0618] In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains comprise an Fc region. In some embodiments the present disclosure provides a method, wherein the first and/or the second and the third engineered immunoglobulin chains comprise an Fc region. Usually the Fc region is selected from the group consisting of IgA, IgE, IgD, IgG, and IgM Fc region.

[0619] In some embodiments the present disclosure provides a method, wherein the first and second engineered immunoglobulin chains comprise an Fc region from the same species, preferably from the same species and isotype, more preferably from the same species, isotype and subclass.

[0620] In some embodiments the present disclosure provides a method, wherein the first and/or the second and the third engineered immunoglobulin chains comprise an Fc region from the same species, preferably from the same species and isotype, more preferably from the same species, isotype and subclass.

[0621] In some embodiments the present disclosure provides a method, wherein the Fc regions of the first and second engineered immunoglobulin chains are are not identical. In some embodiments the present disclosure provides a method, wherein the Fc regions of the first and second engineered immunoglobulin chains have amino acid sequences which are different from each other by one amino acid or by two amino acids or by three amino acids or by four amino acids or by five amino acids or by five to ten amino acids or by ten to thirty amino acids

[0622] In some embodiments the present disclosure provides a method, wherein the hetero-dimeric immunoglobulin or fragment thereof is a full-length antibody.

[0623] In some embodiments the present disclosure provides a method, wherein the hetero-dimeric immunoglobulin or fragment thereof is a full-length antibody which is bispecific.

[0624] In some embodiments the present disclosure provides a bispecific hetero-dimeric immunoglobulin or fragment thereof or a bispecific full-length antibody which binds to antigens selected from the group consisting of HER2, EGPR, CD19 and VLA-2. Preferably the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody is specific to HER2 and EGPR. Equally preferably the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody is specific to CD19 and VLA-2.

[0625] In some embodiments the present disclosure provides a method, wherein wherein at least one additional polypeptide is fused to the first and/or second engineered immunoglobulin chain. Preferably the additional domain is fused to the first engineered immunoglobulin chain.

[0626] Optionally the additional domain can be fused to the first and/or second parent immunoglobulin chain before engineering the parent domain. Suitably the additional domain is selected from the group consisting of FAB, scFv, diabody, domain antibody, pharmacologically active peptide or protein, receptor extracellular domain, CDR, grafted polypeptide and therapeutic engineered protein scaffold.

[0627] In a further aspect the present invention provides a method to engineer a protein-protein interface of a domain of a multidomain protein comprising:

(a) providing a multidomain protein comprising a domain with a protein-protein interface: (b) selecting as a donor domain a naturally occurring immunoglobulin super-family member comprising a domain with a protein-protein interface which is different from the domain of (a); (c) overlaying 3D structures of the domain with the protein-protein interface of (a) and the donor domain with the protein-protein interface of (b); (d) identifying exposed protein-protein interface residues in the overlayed 3D structures of the domain with the protein-protein interface of (a) and the donor domain with the protein-protein interface of (b); e) substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) with amino acid residues at the equivalent 3D structural position from the identified exposed protein-protein interface amino acid residues from the donor domain with the protein-protein interface of (b).

[0628] In some embodiments the present disclosure provides a method, wherein the naturally occurring immunoglobulin superfamily member is a naturally occurring homo-dimer.

[0629] In some embodiments the present disclosure provides a method, wherein the naturally occurring immunoglobulin super-family member is a naturally occurring hetero-dimer. Usually at least two, preferably at least three, more preferably at least four, most preferred at least five, in particular at least six, more particular at least seven amino acid residues are substituted. Preferably the domain with a protein-protein interface of the multidomain protein of (a) is capable to form a homo-dimer, preferably a naturally occurring homo-dimer, with another domain. Preferably the domain with a protein-protein interface of the multidomain protein of (a) is capable to form a hetero-dimer, preferably a naturally occurring hetero-dimer, with another domain. Preferably, the exposed interface residues identified in the immunoglobulin chain and in the selected immunoglobulin super-family members are non essential to the domain core integrity. Preferably, the naturally occurring immunoglobulin super-family member is selected from the TCR constant domain family. More preferably, the donor domain is the TCR constant domain alpha (SEQ ID NO: 1) or the TCR constant domain beta (SEQ ID NO: 2), whereas in an alternative embodiment the cysteine (C) at amino acid position 75 in SEQ ID NO: 2 is substituted with alanine (A) or serine (S). As well preferred as donor domain is a CH3 domain, more preferably a human CH3 domain, in particular a human CH3 domain from IGHG1.

[0630] In a further embodiment the present disclosure provides a method, wherein the donor domain is the TCR constant domain gamma (SEQ ID NO: 33) or the TCR constant domain delta (SEQ ID NO: 32).

[0631] In some embodiments the present disclosure provides a method, wherein the amino acid residues of the protein-protein interface of the parent domain of the first and/or second parent immunoglobulin chain which are substituted are not adjacent.

[0632] Preferably, the identified exposed protein-protein interface amino acid residues of the donor domain with the protein-protein interface of (b) which are used for substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) are selected from the group consisting of K20, V22, T26, Y43, S61, V63, W65 and N67 wherein the amino acid position of each group member corresponds to the amino acid position in SEQ ID NO: 1. Alternatively preferred, the identified exposed protein-protein interface amino acid residues of the donor domain with the protein-protein interface of (b) which are used for substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) are selected from the group consisting of E10, A12, F14, T28, V30, T34, D59, L63, E65, C75, S77, R79 and R81 wherein the amino acid position of each group member corresponds to the amino acid position in SEQ ID NO: 2.

[0633] Preferably, the identified exposed protein-protein interface amino acid residues of the donor domain with the protein-protein interface of (b) which are used for substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) are selected from the group consisting of K4, T6, L22, L24, E28, K29, G53, M56, M63, F65, W67, wherein the amino acid position of each group member corresponds to the amino acid position in SEQ ID NO: 33 Alternatively preferred, the identified exposed protein-protein interface amino acid residues of the donor domain with the protein-protein interface of (b) which are used for substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) are selected from the group consisting of M13, N18, A20, E25, F44, A47, S51, N57, V59, L61, K63, wherein the amino acid position of each group member corresponds to the amino acid position in SEQ ID) NO: 32.

[0634] Preferably, the identified exposed protein-protein interface amino acid residues of the donor domain with the protein-protein interface of (b) which are used for substituting at least one amino acid residue of the identified exposed protein-protein interface amino acid residues of the domain with the protein-protein interface of (a) are selected from the group consisting of Q127, Y129, L131, D136, E137, Q142, S144, T146, K150, K172, T174, V177, D179, F185, Y187, K189, wherein the amino acid position of each group member corresponds to the amino acid position in SEQ ID NO: 12.

[0635] In a further aspect the present invention provides the use of a donor domain of a first and a second member of the naturally occurring immunoglobulin super-family to engineer a hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising a first engineered immunoglobulin chain comprising at least one engineered domain which has a protein-protein interface which is substituted with at least one amino acid residue from the protein-protein interface of the donor domain of the first member of the naturally occurring immunoglobulin super-family and a second engineered immunoglobulin chain comprising at least one engineered domain which has a protein-protein interface which is substituted with at least one amino acid residue from the protein-protein interface of the donor domain of the second member of the naturally occurring immunoglobulin super-family.

[0636] Usually, the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer, preferably a naturally occurring hetero-dimer. Preferably the first and the second member of the naturally occurring immunoglobulin super-family are selected from T cell receptor chains.

[0637] More preferably the donor domain of the first member of the naturally occurring Immunoglobulin super-family is the TCR constant domain alpha (SEQ ID NO: 1) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain beta (SEQ ID NO: 2).

[0638] Equally more preferred the donor domain of the first member of the naturally occurring immunoglobulin super-family is the TCR constant domain delta (SEQ ID NO: 32) and the donor domain of the second member of the naturally occurring immunoglobulin super-family is the TCR constant domain gamma (SEQ ID NO: 33). Equally more preferred, the donor domain of the first and the second member of the naturally occurring immunoglobulin super-family is a CH3 domain.

EXAMPLES

Example 1: Construction of an Immunoglobulin Fc Hetero-Dimer Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains

[0639] This example demonstrates that two human IGHG1 Fc chains (each chain consisting of human hinge (.gamma.1), CH2 and CH3 constant domains; EU residues 221-447) having mutations in the protein-protein interface of their CH3 domains (EU residues 341-447) carefully selected from a subset of the protein-protein interface residues from the human T-cell receptor (TCR) constant domain alpha (GenBank database accession number AAO72258.1 [residues 135-225]; SEQ ID NO: 1; IMGT.RTM. reference TRAC [complete sequence], the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res., 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3): 185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)) for one chain and the human TCR constant domain beta (GenBank database accession number AAA61026.1 [residues 134-261], SEQ ID NO: 2; IMGT.RTM. reference TRBC2 [IMGT.RTM. residues 1.8-124]) for the second chain, assemble into a hetero-dimeric Fc molecule with at least 76% efficacy. TRBC2 is one of two naturally occurring allotypes for the human TCR constant domain beta. Both TRBC1 and TRBC2 can be equivalently used for the purpose to mutate the CH3 domain of IGHG1 Fc chains since there is no difference in the amino acid sequence of their protein-protein interfaces.

[0640] Mutations were derived from the analysis of an overlay of the crystal structure of the LC13 TCR molecule (Kjer-Nielsen L et al., Structure, 10(11):1521-32 (2002)) with the crystal structure of the Fc fragment from human IGHG1 (Krapp S et al., J Mol Biol, 325(5):979-89 (2003)). Both TCR and IGHG1 Fc 3D structures were retrieved from the Protein Data Bank (PDB codes 1KGC and 1H3Y for TRBC2 (SEQ ID NO: 2) and human IGHG1, respectively; www.pdb.org; Bernstein F C et al., Eur J Biochem, 80(2):319-24 (1977)), overlaid with the Coot software (Emsley P and Cowtan K, Acta Crystallogr D Biol Crystallogr, 60(Pt 12 Pt 1):2126-32 (2004)) and further visualized with the Discovery-Studio software from Accelrys (Cambridge, UK). Examination of the protein-protein interface of the overlaid 3D structures of the TCR hetero-dimeric constant domains and the CH3 homo-dimer was used as a starting point for rational design. Several parameters were considered; these included but were not limited to: preservation of the prolines residues, and preservation of amino acid positions involved in the integrity of the domain cores as well as some of the electrostatic contacts from the IGHG1 CH3 homo-dimer; abrogating specific hydrophobic contacts found in the IGHG1 CH3 homo-dimer, and replacing those with selected hydrophobic contacts found in the TCR constant domain hetero-dimer. This analysis led to the design of two subsets of amino acid substitutions, one subset originating from equivalent 3D positions between the protein-protein interface of one subunit of the CH3 homo-dimer and the protein-protein interface of the TCR constant domain alpha, and a second subset originating from equivalent 3D positions between the protein-protein interface of the second subunit of the CH3 homo-dimer and the protein-protein interface of the TCR constant domain beta; each subset creating two new and unique CH3 engineered domain sequences which can heterodimerize.

[0641] The engineered human IGHG1 Fc chain thereof having mutations derived from the protein-protein interface of the human TCR constant domain alpha in the protein-protein interface of its CH3 domain is designated "BT alpha chain" (FIG. 1), while the engineered human IGHG1 Fc chain thereof having mutations derived from the protein-protein interface of the human TCR constant domain beta in the protein-protein interface of its CH3 domain is designated "BT beta chain" (FIG. 1). More specifically, the BT alpha chain consists of an immunoglobulin Fc chain from human IGHG1 having an engineered CH3 domain with the following substitutions: S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, and T411N (SEQ ID NO: 3) (EU numbering); and accordingly, the "BT beta chain" consists of an immunoglobulin Fc chain from human IGHG1 having an engineered CH3 domain with the following substitutions: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V3971L, D399E, F405C, Y407S, K409R, and T411R (SEQ ID NO: 4) (EU numbering).

[0642] The formation of the hetero-dimer Fc can be assessed by SDS-PAGE analysis of samples under non-reducing and reducing conditions. Because the BT alpha chain and the BT beta chain have similar molecular weights, the BT beta chain was fused to a variable light-chain Kappa domain antibody (abbreviated VL, a subfamily Kappal-O12 member derived from the VBASE2 humIGKV115 sequence with the IGKJ*01 junction, SEQ ID NO: 5; Retter I et al., Nucleic Acids Res, 33(Database issue):D671-4 (2005)), to generate a difference in SDS-PAGE mobility and consequently facilitate the identification of hetero-dimer formation (SEQ ID NO: 6) (FIG. 2). Furthermore, to enable confirmation of the presence of the BT alpha chain in the hetero-dimer, a series of six histidine residues was included at its C-terminus (SEQ ID) NO: 7). This hetero-dimeric construct is abbreviated BT alpha His_VL-BT beta.

[0643] To create the BT alpha His and the VL-BT beta chain cDNA coding sequences, a cDNA coding the engineered C143 domain of the BT alpha chain (SEQ ID NO: 8) and the engineered CH3 domain of the BT beta chain were synthesized by GENEART AG (Regensburg, Germany). The BT beta chain was originally synthesized with the F405A mutation, which was later converted to F405C (SEQ ID NO: 9) by standard mutagenesis. Using PCR assembly techniques, each chain had their respective engineered CH3 domain cDNA coding sequence fused downstream of a synthetic cDNA encoding for the human IGHG1 hinge (DKTHTCPPCP) and IGHG1 CH2 constant domain (separately synthesized by GENEART AG). The polyhistidine sequence located at the C-terminus of the BT alpha His chain was included in the anti-sense oligonucleotide during PCR amplification; while the variable Kappa domain antibody located at the N-terminus of the VL-BT beta chain was engineered by fusing the domain cDNA (separately synthesized by GENEART AG) upstream of the BT beta chain cDNA coding sequence using PCR assembly techniques. Finally, the BT alpha His chain and VL-BT beta chain coding DNA sequences (SEQ ID NOs: 10, and 11, respectively) were ligated in independent vectors which are based on a modified pREP4 (Invitrogen, CA, USA) vector carrying CMV promoter and Bovine Growth Hormone poly-adenylation signal. In both chain expression-vectors, secretion was driven by the murine VJ2C leader peptide.

[0644] For transient expression of the BT alpha His_VL-BT beta hetero-dimer, equal quantities of each engineered chains vectors were co-transfected into suspension-adapted HEK-EBNA cells (ATCC-CRL-10852) using Polyethyleneimine (PEI). Typically, 100 ml of cells in suspension at a density of 0.8-1.2 million cells per ml is transfected with a DNA-PEI mixture containing 50 .mu.g of expression vector encoding the BT alpha His chain and 50 g expression vector encoding the VL-BT beta chain. When recombinant expression vectors encoding each engineered chain genes are introduced into the host cells, the hetero-dimeric construct is produced by further culturing the cells for a period of 4 to 5 days to allow for secretion into the culture medium (EX-CELL 293, HEK293-serum-free medium (Sigma, Buchs, Switzerland), supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 .mu.g/ml geneticin). The hetero-dimeric construct was then purified from cell-free supernatant using recombinant Streamline rProtein A media (GE Healthcare Europe GmbH, Glattbrugg, Switzerland), and used for further analysis.

[0645] Transient transfection production yields were above 20 mg/l. The results of a typical SDS-PAGE analysis are shown in FIG. 3. Post protein-A purification the BT alpha His_VL-BT beta hetero-dimer is the main species produced with little homo-dimeric species present (lane 1). Of particular interest is the comparison with the SDS-PAGE mobility of a standard homo-dimeric human IGHG1 Fc (GenBank database accession number AAC82527.1 residues 103-329; SEQ ID NO: 12 (lane 3)), under reducing conditions, the BT alpha His_VL-BT beta hetero-dimer breaks down into the two expected molecular weight bands for the BT alpha His chain and the VL-BT beta chain (lane 2; 25.5 kDa, and 37.2 kDa, respectively; an additional 3 kDa needs to be added to each of these molecular weights to account for chain N-glycosylation at Asn 297) whereas the standard homo-dimeric human IGHG1 Fc (lane 4) collapses into one unique molecular weight band having a similar mobility to the BT alpha His chain. Accordingly, under non-reducing condition, comparison with a standard homo-dimeric human IGHG1 Fc allows identification of traces of BT alpha His homo-dimer present at a very low abundance. Finally aggregates can be also detected at a very low abundance.

[0646] It is possible to assess the proportion of hetero-dimer to homo-dimer in the protein-A purified preparation by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands. Relative ratios of the different species mentioned above were quantified using a FluorChem SP imaging system (Witec AG, Littau, Switzerland) following the protocol provided by the manufacturer. The BT alpha chain His_VL-BT beta chain hetero-dimer represents overall at least 76.1% of the protein-A purified material (FIG. 4A); when omitting traces of aggregates in the relative ratio measurements, the BT alpha chain His_VL-BT beta chain hetero-dimer represents overall at least 83.8% of the protein-A purified material (FIG. 4B).

Example 2: Construction of Immunoglobulin Fc Hetero-Dimer Variants Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains

[0647] T-cell receptor domains are very prone to aggregation and misassociation during folding, and this is often compounded by incorrect disulfide bond formation (Pecorari F et al., J Mol Biol, 285(4):1831-43 (1999)). Mutation of the unpaired cysteine in the TCR constant domain beta has been shown to facilitate the production of TCR molecules in E. coli (Willcox B E et al., Protein Sci, 8(11):2418-23 (1999)). In addition, this unpaired cysteine has been mutated in several TCR molecule crystal structures. Mutations of C40A and C405S in the BT beta chain coding DNA sequences were derived from the analysis of an overlay of the crystal structure of the Fc fragment from human IGHG1 (Krapp S et al., J Mol Biol, 325(5):979-89 (2003)) with the crystal structure of the CD1d-.alpha.-GalCer-specific TCR molecule 5E (PDB code 2CDF, Gadola S D et al., J Exp Med, 203(3):699-710 (2006)) and the crystal structure of the T cell receptor (TCR) HA 1.7 (PDB code IJ8H, Hennecke J and Wiley D C, J Exp Med, 195(5):571-81 (2002)). Since the engineered CH3 domain of the BT beta chain was originally synthesized with the F405A mutation, this construct was directly used to build a new VL-BT beta chain identical to the VL-BT beta chain described in example 1 except for the introduction of the F405A mutation. The VL-BT beta chain having the F405A mutation was then modified to create a second VL-BT beta chain having the F405S mutation using standard mutagenesis techniques. These two new chains identical to the VL-BT beta chain described in example 1 expect for their F405A and F405S mutations respectively, are referred herein as "VL-BT beta F405A chain" or "VL-BT beta F405A" and "VL-BT beta F405S chain" or "VL-BT beta F405S".

[0648] Transient expression of the BT alpha His_VL-BT beta F405S hetero-dimer and BT alpha His_VL-BT beta F405A hetero-dimer were performed as described in example 1. Production yields for the BT alpha His_VL-BT beta F405A hetero-dimer were similar to the BT alpha His_VL-BT beta hetero-dimer (22 mg/l each). For the BT alpha His_VL-BT beta F405S hetero-dimer production yields of 37 mg/l were obtained. To assess the quality of the BT alpha His_VL-BT beta F405S hetero-dimer formation over the BT alpha His_VL-BT beta hetero-dimer described in example 1, both BT hetero-dimers were run on the same SDS-PAGE gel (FIG. 3, lane 1 (BT alpha His_VL-BT beta) and 5 (BT alpha His_VL-BT beta F405S)). Post protein-A purification the BT alpha His_VL-BT beta F405S hetero-dimer is the main species produced, however when compared to the BT alpha His_VL-BT beta hetero-dimer described in example 1, significantly higher amounts of monomeric species as well as VL-BT beta F405S homo-dimer species were observed, indicating a higher hetero-dimeric assembly efficacy of the naturally occurring F405C substitution over the mutated F405S substitution. Similarly to the BT alpha His_VL-BT beta hetero-dimer, under reducing conditions, the BT alpha His_VL-BT beta F405S hetero-dimer breaks down into the two expected molecular weight bands for the BT alpha His chain and the VL-BT beta F405S chain (lane 6).

[0649] The results of a typical SDS-PAGE analysis for the BT alpha His_VL-BT beta F405A hetero-dimer are shown in FIG. 5. Post protein-A purification the BT alpha His_VL-BT beta F405A hetero-dimer is the main species produced with little homo-dimeric species present (lane 1). Similarly to the BT alpha His_VL-BT beta hetero-dimer, under reducing conditions, the BT alpha His_VL-BT beta F405A hetero-dimer breaks down into the two expected molecular weight bands for the BT alpha His chain and the VL-BT beta F405A chain (lane 2). Accordingly, under non-reducing condition, comparison with a standard homo-dimeric human IGHG1 Fc (lane 3: non-reducing conditions, lane 4: reducing conditions) allows identification of traces of BT alpha His homo-dimer present at a very low abundance. Finally traces of VL-BT beta F405A homo-dimer and monomer as well as aggregates can be also detected at a very low abundance.

[0650] To further demonstrate the identity of BT alpha His_VL-BT beta F405A hetero-dimer, the protein-A purified material was loaded on Ni.sup.2+ affinity sepharose (GE Healthcare Europe GmbH, Glattbrugg, Switzerland), a chromatographic step which is specific for species containing the BT alpha His chain due to its polyhistidine sequence. After a wash step, bound and eluted fractions from the Ni.sup.2+ affinity step were pooled and loaded onto protein-L affinity agarose (GenScript USA Inc., Piscataway, N.J., USA), a superantigen affinity resin which specifically select for species having the variable light-chain Kappa domain antibody, i.e. the VL-BT beta F405A chain. Outputs of the above purification steps were then assessed by Western blotting with an antibody against the polyhistidine sequence labeled with horse-radish peroxidase, thereby detecting the BT alpha His chain. The blot was stained using Sigma Fast 3,3'-Diaminobenzidine precipitating substrate (Sigma, Buchs, Switzerland). FIG. 6 shows that the BT alpha His chain is consistently detected before, and after Ni.sup.2+ sepharose but also and more importantly after protein-L agarose chromatography, demonstrating the identity of the hetero-dimer.

[0651] It is possible to assess the proportion of hetero-dimer to homo-dimer in the protein-A purified preparation by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands. Relative ratios of the different species mentioned above were quantified using a FluorChem SP imaging system (Witec AG, Littau, Switzerland) following the protocol provided by the manufacturer. The BT alpha chain His_VL-BT beta chain hetero-dimer represents overall at least 80.7% of the protein-A purified material (FIG. 7A); when omitting traces of aggregates and VL-BT beta F405A chain monomer in the relative ratio measurements, the BT alpha chain His_VL-BT beta F405A chain hetero-dimer represents overall at least 86.9% of the protein-A purified material (FIG. 7B).

[0652] Protein stability is a crucial issue for the development of recombinant antibody therapeutics. A poor stability can affect the ability of an antibody to fold when expressed in various cell types (Garber E and Demarest S J, Biochem BiophyS Res Commun, 355(3):751-7 (2007)), and lead to a fraction of the clinical material to be misfolded, non functional and/or prone to aggregate, and thus potentially immunogenic (Chirino A J et al., Drug Discov Today, 9(2):82-90 (2004)). To assess the intrinsic stability of the hetero-dimeric Fc fragment without any interference from an additional protein domain, a BT beta chain without variable kappa domain (this chain was engineered similarly to the other BT beta chains but had a C-terminal HA tag (YPYDVPDYA) and no VL domain, abbreviated BT beta (F405A) HA chain; SEQ ID NO: 17) was co-expressed with a BT alpha His chain. The resulting hetero-dimeric molecule; B31 alpha His_BT beta (F405A) HA was purified as above and analyzed using differential scanning calorimetry (DSC). The calorimetric measurements were carried out on a VP-DSC differential scanning microcalorimeter (GE Healthcare Europe GmbH, Glattbrugg, Switzerland). The cell volume was 0.128 ml, the heating rate was 1.degree. C./min, and the excess pressure was kept at 64 p.s.i. The protein was used at a concentration of 2 mg/ml in PBS (pH 7.4). The molar heat capacity of the protein was estimated by comparison with duplicate samples containing identical buffer from which the protein had been omitted. The partial molar heat capacities and melting curves were analyzed using standard procedures. Thermograms were baseline corrected and concentration normalized before being further analyzed using a Non-Two State model in the Origin (v7.0) software (OriginLab Corporation, Northampton, Mass., USA; provided by GE Healthcare Europe GmbH, Glattbrugg, Switzerland).

[0653] The expected melting profiles for the human IgG subclasses are known (Garber E and Demarest S J, Biochem Biophys Res Commun, 355(3):751-7 (2007)), and all profiles have been shown to contain three unfolding transitions corresponding to the independent unfolding of the CH2, CH3, and FAB domains. Of the four human IgG subclasses, IGHG1 has the most stable CH3 domain (.about.85.degree. C.); while other subclasses CH3 domains are less stable, although none are known to melt below 70.degree. C. Similarly, all subclasses are known to have a melting temperature of .about.70.degree. C. for the CH2 domain (Garber E and Demarest S J, Biochem Biophys Res Commun, 355(3):751-7 (2007)). Overall no melting transition is expected to be lower than 70.degree. C. for a stable antibody Fc fragment (hinge-CH2-CH3). FIG. 7C shows a thermogram of the BT alpha His_BT beta (F405A) HA hetero-dimer exhibiting a single sharp transition at 70.degree. C., accounting for the melting transitions of both the CH2 domain and the engineered CH3 domain. It is important to note that no sharp decrease after the heat absorption peak was recorded, indicating that no precipitation or aggregate formation occurred after thermal unfolding as expected for a stable antibody Fc fragment (Liu H et al., Immunol Lett, 106(2):144-53 (2006)). Furthermore, the hetero-dimeric Fc fragment has stability similar to the naturally occurring Fc fragments since the melting transitions of its domains are similar to the ones observed for the human IGHG4 Fc fragment where both the CH2 domain and the CH3 domain melt at approximately 70.degree. C. (Garber E and Demarest S J, Biochem Biophys Res Commun, 355(3):751-7 (2007)). In conclusion, this thermal unfolding study shows that the hetero-dimeric Fc fragment with engineered protein-protein interfaces based on the TCR alpha and beta constant domains is stable and suitable for building further therapeutic hetero-dimeric immunoglobulins.

Example 3: Construction of Immunoglobulin Fc Hetero-Dimer Variants Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains with Minimal Substitutions

[0654] To identify pairs of BT alpha and beta chains that will form hetero-dimers over the formation of possible homo-dimers with a reduced number of amino acid substitutions originating from the key protein-protein interface residues from the human T-cell receptor (TCR) constant domain alpha (GenBank database accession number AAO72258.1, residues 135-225; SEQ ID NO: 1; IMGT.RTM. reference TRAC [complete sequence], the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res. 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3): 185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)) for one chain and the human TCR constant domain beta (GenBank database accession number AAA61026.1 [residues 134-261], SEQ ID NO: 2; IMGT.RTM. reference TRBC2 [IMGT.RTM. residues 1.8-124]) for the second chain, two or more of the substitutions described in example 1 and 2 were gradually introduced in each chain.

[0655] There are multiple subsets of amino acid substitutions originating from the key protein-protein interface residues from the human TCR constant domain alpha and beta that can be chosen to alternate between the TCR constant domain sequence and the human IGHG1 Fc sequence in the BT alpha and beta chains, respectively. The choice of residues for each considered subset is based on structural considerations, for example, protein-protein surface interaction calculations. All substitution subsets were engineered by mutating the human IGHG1 Fc DNA coding sequence (SEQ ID NO: 12) using standard molecular biology techniques (example 1). The resulting variants were then ligated independently into the modified pREP4 vector mentioned previously and co-transfected into HEK293-EBNA cells as described in example 1. Protein production was also according to example 1. Each subset of substitutions was assessed for its rate of hetero-dimerization using a combination of SDS-PAGE analysis of samples under non-reducing and reducing conditions, and scanning densitometry (as stated in example 1).

[0656] Because the engineered chains have close molecular weights, similarly to example 1, the BT beta chain variants were fused to a variable light-chain kappa domain antibody (abbreviated VL, a subfamily Kappa I-O12 member derived from the VBASE2 humIGKV115 sequence with the IGKJ1*01 junction, SEQ ID NO: 5; Retter I. et al, 2005, Nucleic Acids Res., 33, Database issue D671-D674) to generate a difference in SDS-PAGE mobility and consequently facilitate the identification of hetero-dimer formation. To differentiate these chains from example 1, and since these BT chains have identical amino acid sequence to the human IGHG1 Fc sequence except for the specific TCR alpha and beta constant domain based substitutions which have been introduced by site-directed mutagenesis, the BT alpha and VL-BT beta chains are herein abbreviated Fc (BTA) chain and VL-Fc (BTB) chain, respectively. For each chain, the specific substitutions which have been introduced into the human IGHG1 Fc sequence are indicated using the EU numbering. The result of these expression experiments, in terms of yield and hetero-dimerization rate are summarized in Table 1. Two to six substitutions were made in each chain.

[0657] The results of a typical SDS-PAGE analysis are shown in FIG. 8. Post protein-A purification the hetero-dimer is the main species produced with little homo-dimeric species present (lane1). Under reducing conditions the hetero-dimer breaks down into the two expected molecular weight bands for the engineered Fc (BTA) chain and the other engineered VL-Fc (BTB) chain (lane 2; .about.25 kDa and .about.37 kDa, respectively). Transient transfection production yields were up to 50 mg/l while the best hetero-dimerization rate was 90%.

[0658] The best combination of substitution subsets was: Fc (BTA)-S364K-T366V-K370T-K392Y-K409W-T411N_VL-Fc (BTB)-F405A-Y407S (SEQ ID NOs: 23 and 24) with a 90% hetero-dimerization rate (FIG. 9) and a transient transfection yield of 30 mg/L The minimal number of substitutions tested in each chain was two with a 64% hetero-dimerization rate and a transient transfection yield of 35 mg/l. To compare the hetero-dimerization technology described herein with existing methods, a "knob-into-hole" Fc variant (Merchant A M et al., Nat Biotechnol, 16(7):677-81 (1998)) with the portion encompassing the "hole" fused to a variable light-chain kappa domain antibody (VL, SEQ ID NO: 5) was created (abbreviated Fc-T366W_VL-Fc-T366S-L368A-Y407V; EU numbering: SEQ ID NOs: 30 and 31). In side by side expression experiments, the "knob-into-hole" based molecule had only a 66% hetero-dimerization rate (data not shown).

TABLE-US-00001 TABLE 1 Production yields of minimal BT chains. Hetero-dimer formation percentage is indicated in brackets and represents the percentage of hetero-dimer in the protein-A purified material when omitting impurities in the relative ratio measurements. VL-Fc (BTB) chain F405A F405A Y407S F405A F405A Y407S K409R Y407S K409R Fc (BTA) Y407S K409R K409R T411R K409R T411R chain SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 Y407V 35 mg/l 11 mg/l 39 mg/l 41 mg/l 45 mg/l 28 mg/l K409W (64) (32) (30) (36) (47) (36) SEQ ID NO: 18 K392Y 35 mg/l 46 mg/l 30 mg/l 18 mg/l 35 mg/l 29 mg/l K409W (52) (46) (46) (14) (50) (30) SEQ ID NO: 19 T366V 51 mg/l 50 mg/l 27 mg/l 26 mg/l 26 mg/l 26 mg/l K409W (57) (46) (49) (26) (49) (34) SEQ ID NO: 20 Y407V 18 mg/l 20 mg/l 5 mg/l 25 mg/l 4 mg/l 21 mg/l K409W (50) (25) (25) (not (52) (53) T411N quantifiable) SEQ ID NO: 21 F405S 8 mg/l 7 mg/l 8 mg/l 12 mg/l 4 mg/l 29 mg/l Y407V (7) (4) (5) (not (15) (9) K409W quantifiable) T411N SEQ ID NO: 22 S364K 30 mg/l 5 mg/l 7 mg/l 9 mg/l 10 mg/l 42 mg/l T366V (90) (60) (74) (32) (72) (88) K370T K392Y K409W T411N SEQ ID NO: 23

Example 4: Construction of an Immunoglobulin Fc Hetero-Dimer Having a CH3-CH3 Protein-Protein Interface Based on the Human .delta./.gamma. T Cell Receptor Constant Domains

[0659] This example demonstrates that two human IGHG1 Fc chains (each chain consisting of human hinge (.gamma.1), CH2 and CH3 constant domains; EU residues 221-447) having mutations in the protein-protein interface of their CH3 domains (EU residues 341-447) carefully selected from a subset of the protein-protein interface residues from the human T-cell receptor (TCR) constant domain delta (GenBank database accession number AAA61125.1 [residues 135-221]; SEQ ID NO: 32; IMGT.RTM. reference TRDC [IMGT.RTM. residues 1.7-120], the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al. Dev Comp Immunol, 29(3): 185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)) for one chain and the human TCR constant domain gamma (GenBank database accession number AAA61110.1 [residues 145-245], SEQ ID NO: 33; IMGT.RTM. reference TRGC1 [IMGT.RTM. residues 1.1-124]) for the second chain, assemble into a hetero-dimeric Fc molecule with at least 52% efficacy. TRGC1 and TRDC are naturally occurring isotypes. TRGC1 is one of two naturally occurring allotypes for the human TCR constant domain gamma. Both TRGC1 and TRGC2 (termed 2.times. or 3.times.) can be equivalently used for the purpose to mutate the CH3 domain of IGHG1 Fc chains since there is no difference in the amino acid sequence of their protein-protein interfaces.

[0660] Mutations were derived from the analysis of an overlay of the crystal structure of the the G115 .gamma..delta.TCR molecule (Allison T J et al., Nature, 411 (6839):820-4 (2001)) with the crystal structure of the Fc fragment from human IGHG1 (Krapp S et al., J Mol Biol, 325(5):979-89 (2003)). Both TCR and IGHG1 Fc 3D structures were retrieved from the Protein Data Bank (PDB codes 1HXM and 1H3Y for human TCR and human IGHG1, respectively; www.pdb.org; Bernstein F C et al, Eur J Biochem, 80(2):319-24 (1977)), overlaid with the Coot software (Emsley P and Cowlan K, Acta Crystallogr D Biol Crystallogr, 60(Pt 12 Pt 1):2126-32 (2004)) and further visualized with the Discovery-Studio software from Accelrys (Cambridge, UK). Examination of the protein-protein interfaces of the overlaid 3D structures of the TCR hetero-dimeric constant domains and the CH3 homo-dimer was used as a starting point for rational design. Several parameters were considered; these included but were not limited to: the preservation of the prolines residues and the preservation of amino acid positions involved in the integrity of the domain cores as well as some of the electrostatic contacts from the IGHG1 CH3 homo-dimer; abrogating specific hydrophobic contacts found in the IGHG1 CH3 homo-dimer, and replacing those with selected hydrophobic contacts found in the TCR constant domain hetero-dimer. This analysis led to the design of two subsets of amino acid substitutions, one subset originating from equivalent 3D positions between the protein-protein interface of one subunit of the CH3 homo-dimer and the protein-protein interface of the TCR constant domain delta, and a second subset originating from equivalent 3D positions between the protein-protein interface of the second subunit of the CH3 homo-dimer and the protein-protein interface of the TCR constant domain gamma; each subset creating two new and unique CH3 engineered domain sequences which can hetero-dimerize.

[0661] The engineered human IGHG1 Fc chain thereof having mutations derived from the protein-protein interface of the human TCR constant domain delta in the protein-protein interface of its CH3 domain is designated "BT delta chain", while the engineered human IGHG1 Fc chain thereof having mutations derived from the protein-protein interface of the human TCR constant domain gamma in the protein-protein interface of its CH3 domain is designated "BT gamma chain". More specifically, the BT delta chain consists of an immunoglobulin Fc chain from human IGHG1 having an engineered CH3 domain with the following substitutions: L351M, S364N, T366A, G371E, K392F, T394A, D399S, F405N, Y407V, K409L, and T411K (SEQ ID NO: 34) (EU numbering); and accordingly, the "BT gamma chain" consists of an immunoglobulin Fc chain from human IGHG1 having an engineered CH3 domains with the following substitutions: Q347K, Y349T, S364T, T366L, K370E, G371K, T394G, V397M, F405M, Y407P, K409W (SEQ ID NO: 35) (EU numbering).

[0662] The formation of the Fc hetero-dimer can be assessed by SDS-PAGE analysis of samples under non-reducing and reducing conditions. Because the BT delta chain and the BT gamma chain have similar molecular weights, the BT gamma chain was fused to a variable light-chain kappa domain antibody (abbreviated VL, a subfamily Kappa I-O12 member derived from the VBASE2 humIGKV115 sequence with the IGKJ1*01 junction. SEQ ID NO: 5; Retter I et al., Nucleic Acids Res, 33(Database issue):D671-4 (2005)) (VL-BT gamma, SEQ ID NO: 36), to generate a difference in SDS-PAGE mobility and consequently facilitate the identification of hetero-dimer formation. The resulting hetero-dimeric construct is abbreviated BT delta_VL-BT gamma.

[0663] To create the BT delta and the VL-BT gamma chain cDNA coding sequences, a cDNA coding the engineered CH3 domain of the BT delta chain (SEQ ID NO: 37) and the engineered CH3 domain of the BT gamma chain (SEQ ID NO: 38) were synthesized by GENEART AG (Regensburg, Germany). Using PCR assembly techniques, each chain had their respective engineered CH3 domain cDNA coding sequence fused downstream of a synthetic cDNA coding for the human hinge IGHG1 (DKTHTCPPCP) and CH2 constant domain (separately synthesized by GENEART AG). The variable kappa domain antibody located at the N-terminus of the VL-BT gamma chain was engineered by fusing the domain cDNA (separately synthesized by GENEART AG) upstream of the BT gamma chain cDNA coding sequence using PCR assembly techniques.

[0664] The resulting variants were then ligated independently into the modified pREP4 vector mentioned previously and cotransfected into HEK293-EBNA cells as described in example 1. Protein production was also according to example 1. Transient transfection yields were up to 60 mg/l. The results of a typical SDS-PAGE analysis are shown in FIG. 10.

[0665] To assess the proportion of hetero-dimer to homo-dimer in the protein-A purified material, the relative ratios of the different species were quantified by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands according to the procedure described in example 1. The BT delta_VL-BT gamma hetero-dimer represents at least 52.1% of the protein-A purified material (FIG. 11A): when omitting traces of aggregates and monomer in the relative ratio measurements, the BT delta_VL-BT gamma hetero-dimer represents at least 57.5% of the protein-A purified material (FIG. 11B).

Example 5: Construction of a Monovalent Immunoglobulin with a Hetero-Dimeric Fc Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains, and a FAB Fragment Having a CH1-CK Protein-Protein Interface Based on the Hetero-Dimeric .delta./.gamma. T Cell Receptor Constant Domains

[0666] This example demonstrates that at least three human immunoglobulin chains: one chain consisting of a heavy chain variable domain fused to the human heavy chain constant domains CH1 (.gamma.1), hinge (.gamma.1), CH2 (.gamma.1) and engineered CH3 domain from the BT alpha or BT beta chain, one BT alpha or one BT beta chain, and one chain consisting of a light chain variable domain fused to the human CK light chain constant domain wherein the protein-protein interface of the CH1 (.gamma.1) (IGHG1, EU residues 118-215) and C.kappa. (IGKC, EU residues 108-214) domains have been carefully substituted at selected positions with a subset of the protein-protein interface residues from the naturally occurring human TCR constant domain delta (Genbank database accession number AAA61125.1 [residues 135-221]; SEQ ID NO: 32; IMGT.RTM. reference TRDC [IMGT.RTM. residues 1.7-120], the international ImMunoGencTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acid Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acid Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)), and the naturally occurring human TCR constant domain gamma (GenBank database accession number AAA61110.1 [residues 145-245]; SEQ ID NO: 33; IMGT.RTM. reference TRGC1 [IMGT.RTM. residues 1.1-124]) respectively, assemble into a FAB engineered hetero-dimeric immunoglobulin molecule with at least 50% efficacy.

[0667] Mutations were derived from the analysis of an overlay of the crystal structure of the TCR molecule with the crystal structure of a human FAB (.gamma.1) fragment. Both TCR and human FAB (.gamma.1) figment 3D structures were retrieved from the Protein Data Bank (PDB code 1HXM, and 1VGE, respectively; Allison T J et al., Nature, 411(6839):820-4 (2001); Chacko S et al., J Biol Chem. 271(21):12191-8 (1996)), overlaid with the Coot software (Emsley P and Cowtan K, Acta Crystallogr D Biol Crystallogr, 60(Pt 12 Pt 1):2126-32 (2004)) and further visualized with the Discovery-Studio software from Accelrys (Cambridge, UK). Examination of the protein-protein interfaces of the overlaid 3D structures of the .delta./.gamma. TCR hetero-dimeric constant domains and the CH1 (.gamma.1)-C.kappa. hetero-dimer was used as a starting point for rational design. Several parameters were considered; these included but were not limited to: the preservation of the proline residues and the preservation of amino acid positions involved in the integrity of the domain cores as well as some of the electrostatic contacts between the CH1 (.gamma.1), and the C.kappa. domains; abrogating specific hydrophobic contacts found in the CH1 (.gamma.1)-C.kappa. hetero-dimer, and replacing those with selected hydrophobic contacts found in the TCR constant domain gamma-delta hetero-dimer. This analysis led to the design of two subsets of amino acid substitutions, one subset originating from equivalent 3D positions between the protein-protein interface of the CH1 (.gamma.1) domain and the protein-protein interface of the TCR constant domain delta, and a second subset originating from equivalent 3D positions between the protein-protein interface of the CK domain and the protein-protein interface of the TCR constant domain gamma; thereby creating two engineered, novel and unique domains which can still hetero-dimerize but differently from the naturally occurring CH1 (.gamma.1)-C.kappa. hetero-dimeric domain pair.

[0668] An engineered heavy chain of a FAB fragment comprising a variable heavy chain domain, and a human CH1 (.gamma.1) domain having mutations derived from the protein-protein interface of the human TCR constant domain delta in its protein-protein interface is designated "VH-CH1 delta" (FIG. 12), while an engineered kappa light chain comprising a variable light chain domain, and a human C.kappa. domain having mutations derived from the protein-protein interface of the human TCR constant domain gamma in its protein-protein interface is designated "VL-CK gamma" (FIG. 12). More specifically, a VH-CH1 delta consists of an engineered heavy chain FAB fragment having an engineered CH1 (.gamma.1) domain with the following substitutions: L128M, A141N, G143A, D148E, H168F, F170A, Q175S, S181N, S183V, V185L, T187K (SEQ ID NO: 39) (EU numbering); and accordingly, a VL-CK gamma consists of an engineered kappa light chain having an engineered C.kappa. domain with the following substitutions: S114K, F116T, S131T1, V133L, N137E, N138K, S162G, T164M, S174M, S176F, T178W (SEQ ID NO: 40) (EU numbering).

[0669] In this example, the variable heavy chain and light chain domains from a humanized anti human CD19 antibody disclosed in the PCT Publication NO: WO 2010/095031 (abbreviated VH(anti-hCD19), and VL(anti-hCD19)) were used as inputs to build the VH-CH1 delta and the VL-CK gamma chains, respectively. The combination of a VH-CH1 delta chain fused upstream of a previously described BT beta chain (examples 1-3) (note that in this example, the chain had a C-terminal c-myc tag (EQKLISEEDLN), abbreviated BT beta c-myc chain) results in a newly engineered immunoglobulin heavy chain which once co-expressed with the newly engineered VL-CK gamma light chain described above, and a BT alpha His chain described in examples 1-3, creates a new type of hetero-dimeric monovalent (i.e. having one specificity for one antigen) immunoglobulin molecule having a novel FAB fragment wherein the CH1 (.gamma.1) and CK constant domains have novel protein-protein interfaces based on the TCR delta and TCR gamma constant domain protein-protein interfaces, respectively, and a Fc fragment with hetero-dimeric engineered CH3 domains based on the TCR alpha and beta constant domains (examples 1-3). This novel immunoglobulin molecule is abbreviated VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma (SEQ ID NOs: 41, 7, and 42).

[0670] To create the VH(anti-hCD19)-CH1 delta-BT beta c-myc chain and the VL(anti-hCD19)-CK gamma chain cDNA coding sequences (SEQ ID NOs: 43 and 44, respectively), a unique cDNA encompassing both the CH1 delta domain sequence (SEQ ID NO: 45) and the CK gamma domain sequence (SEQ ID NO: 46) was synthesized by GENEART AG (Regensburg, Germany). Using PCR, each chain was amplified individually, and the respective engineered constant domain cDNA coding sequences were fused downstream of a synthetic cDNA coding for their respective variable domains (separately synthesized by GENEART AG). The resulting VH(anti-hCD19)-CH1 delta cDNA fragment was then further fussed upstream of a BT beta F405A chain having a C-terminal c-myc tag to generate the final VH(anti-hCD19)-CH1 delta-BT beta c-myc chain. Subsequently, the two chains were then ligated independently in the modified pREP4 vector described in example 1. The BT alpha His chain originated from example 1 (SEQ ID NO: 10).

[0671] For transient expression of the VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule, equal quantities of three engineered chain vectors were co-transfected into suspension-adapted HEK293-EBNA cells as described previously (example 1). The FAB engineered hetero-dimeric construct was then purified from the cell-free supernatant using protein-A affinity chromatography and used for further analysis (procedure according to example 1). The correct assembly of the FAB engineered hetero-dimer can be assessed by SDS-PAGE analysis of samples under non-reducing conditions, since the correctly assembled molecule is expected to have a different SDS-PAGE mobility from the possible homo-dimeric species.

[0672] Transient transfection yields were above 16 mg/l. The results of a typical SDS-PAGE analysis are shown in FIG. 13. Post protein-A purification the VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule (lane1) is the main species produced (A) with some homo-dimeric species (B) and some half-molecules (C) present.

[0673] It is possible to assess the proportion of hetero-dimer to homo-dimer in the protein-A purified preparation by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands (procedure according to example 1). FIG. 14 shows that the VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule represents at least 50% of the protein-A purified material.

[0674] The thermal stability of the VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule was assessed using calorimetric measurements (DSC) (see example 2 for methods). Monoclonal antibodies have melting profiles which are characteristic of their isotypes, and which usually combine transitions for the FAB fragment, the CH2 and the CH3 domains (see example 2); within an antibody melting profile, the specific transition for the FAB fragment can easily be identified (Garber, E, and S. J. Demarest, 2007, Biochem Biophys Res Commun., 355(3): 751-7). FIG. 15 shows an example of a thermal unfolding experiment for the VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule: two overlapping sharp transitions at 70.7.degree. C., and .about.75.degree. C. are observed, accounting for the melting transitions of the engineered FAB fragment, the CH2 domains, and the engineered CH3 domains. Since the BT alpha His_BT beta (F405A) HA hetero-dimeric Fc displayed a single sharp melting transition at 70.degree. C. in previous measurements (example 2), it is possible to conclude that the melting transition of the engineered FAB fragment of the VH(anti-hCD19)-CH1 delta-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK gamma molecule has a mid-point of .about.75.degree. C. It is important to note that no sharp decrease after the heat absorption peak was recorded, indicating that no precipitation or aggregate formation occurred after thermal unfolding (Liu H et al., Immunol Lett, 106(2):144-53 (2006)). In conclusion, this thermal denaturation study shows that the novel FAB fragment with engineered protein-protein interfaces based on the TCR delta and gamma constant domains is stable and suitable for building further therapeutic hetero-dimeric immunoglobulins; more precisely, immunoglobulin molecules comprising two novel hetero-dimeric pairs of constant domains, one pair having engineered protein-protein interfaces based on the human T-cell receptor constant domain alpha and beta, and the other pair having engineered protein-protein interfaces based on the human T-cell receptor constant domain delta and gamma.

Example 6: Construction of a Monovalent Immunoglobulin with a Hetero-Dimeric Fc Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains, and a FAB Fragment Having a CH1-CK Protein-Protein Interface Based on the Homo-Dimeric IGHG1 CH3 Constant Domains

[0675] This example demonstrates that at least three human immunoglobulin chains: one chain consisting of a heavy chain variable domain fused to the human heavy chain constant domains CH1 (.gamma.1), hinge (.gamma.1), CH2 (.gamma.1) and engineered CH3 domain from the BT alpha or BT beta chain, one BT alpha or one BT beta chain, and one chain consisting of a light chain variable domain fused to the human C.kappa. light chain constant domain wherein the protein-protein interface of the CH1 (.gamma.1) (IGHG1, EU residues 118-215) and C.kappa. (IGKC, EU residues 108-214) domains have been carefully substituted at selected positions with a subset of the protein-protein interface residues from the human CH3 (1) constant domain (IGHG1, EU residues 341-447; SEQ ID NO: 47), assemble into a FAB engineered hetero-dimeric immunoglobulin molecule with at least 51.9% efficacy.

[0676] Mutations were derived from the analysis of an overlay of the crystal structure of a Fc (.gamma.1) fragment with the crystal structure of a human FAB (.gamma.1) fragment. Both Fc (.gamma.1) fragment and human FAB (.gamma.1) fragment 3D structures were retrieved from the Protein Data Bank (PDB code 1H3U, and 1VGE, respectively; Krapp S et al., J Mol Biol, 325(5):979-89 (2003), Chacko S et al., J Biol Chem, 271(21):12191-8 (1996)), overlaid with the Coot software (Emsley P and Cowtan K, Acta Crystallogr D Biol Crystallogr, 60(Pt 12 Pt 1):2126-32 (2004)) and further visualized with the Discovery-Studio software from Accelrys (Cambridge, UK). Examination of the protein-protein interfaces of the overlaid 3D structures of the homo-dimeric CH3 (.gamma.1) constant domain pair and the CH1 (.gamma.1)-C.kappa. hetero-dimer was used as a starting point for rational design. Several parameters were considered; these included but were not limited to: the preservation of the proline residues and the preservation of amino acid positions involved in the integrity of the domain cores as well as some of the electrostatic contacts from the CH1 (.gamma.1)-C.kappa. hetero-dimer, abrogating specific hydrophobic contacts found in the CH1 (.gamma.1)-C.kappa. hetero-dimer, and replacing those with selected hydrophobic contacts found in the homo-dimeric CH3 constant domain pair. This analysis led to the design of two subsets of amino acid substitutions, one subset originating from equivalent 3D positions between the protein-protein interface of the CH1 (.gamma.1) constant domain and the protein-protein interface of the human CH3 (.gamma.1) constant domain, and a second subset originating from equivalent 3D positions between the protein-protein interface of the C.kappa. domain and the protein-protein interface of the human CH3 (.gamma.1) constant domain; thereby creating two engineered, novel and unique domains which can still hetero-dimerize but differently from the naturally occurring CH1 (.gamma.1)-C.kappa. hetero-dimeric domain pair.

[0677] An engineered heavy chain of a FAB fragment comprising a variable heavy chain domain, and a human CH1 (.gamma.1) domain having mutations derived from the protein-protein interface of the C3 (.gamma.1) constant domain in its protein-protein interface is designated "VH-CH13" (FIG. 16), while an engineered kappa light chain comprising a variable light chain domain, and an engineered human C.kappa. constant domain having mutations derived from the protein-protein interface of the CH3 (.gamma.1) constant domain in its protein-protein interface is designated "VL-CK3" (FIG. 16). More specifically, a VH-CH13 chain consists of an engineered heavy chain FAB fragment having an engineered CH1 (.gamma.1) domain with the following substitutions: S124Q, F126Y, K133D, S134E, T139Q, A141S, G143T, H168K, F170T, Q175D, S181F, S183Y, V185K (SEQ ID NO: 48) (EU numbering); and accordingly, a VL-CK3 domain consists of an engineered light chain FAR fragment having an engineered C.kappa. domain with the following substitutions: S114Q, F116Y, F118L, E123D, Q124E, V133T, N137K, Q160K, S162T, T164V, S174F, S176Y, T178K (SEQ ID NO: 49) (EU numbering).

[0678] In this example, the variable heavy chain and light chain domains from a humanized anti human CD19 antibody disclosed in the PCT Publication NO: WO 2010/095031 (abbreviated VH(anti-hCD19), and VL(anti-hCD19)) were used as inputs to build the VH-CH13 and the VL-CK3 chains, respectively. The combination of a VH-CH13 chain fused upstream of a previously described BT beta chain (note that in this example, the chain had a C-terminal c-myc tag (EQKLISEEDLN), abbreviated BT beta c-myc chain), results in a newly engineered immunoglobulin heavy chain which once co-expressed with the newly engineered VL-CK3 light chain described above, and a BT alpha His chain described in examples 1-3, creates a new type of hetero-dimeric monovalent (i.e. having one specificity for one antigen) immunoglobulin molecule having a novel FAB fragment wherein the CH1 (.gamma.1) and C.kappa. constant domains have novel protein-protein interfaces based on the CH3 (.gamma.1) constant domain homo-dimeric protein-protein interface, and a Fc fragment with hetero-dimeric engineered CH3 domains based on the TCR alpha and beta constant domains (see examples 1-3). This novel immunoglobulin molecule is abbreviated VH(anti-hCD19)-CH3-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 (SEQ ID NOs: 50, 7, and 51).

[0679] To create the VH(anti-hCD19)-CH3-BT beta c-myc chain and the VL(anti-hCD19)-CK3 chain cDNA coding sequences (SEQ ID NOs: 52 and 53, respectively), a unique cDNA encompassing both the CH13 domain sequence (SEQ ID NO: 54) and the CK3 domain sequence (SEQ ID NO: 55) was synthesized by GENEART AG (Regensburg, Germany). Using PCR, each chain was amplified individually, and the respective engineered constant domain cDNA coding sequences were fused downstream of a synthetic cDNA coding for their respective variable domains (separately synthesized by GENEART AG). The resulting VH(anti-hCD19)-CH13 DNA fragment was further fused upstream of a BT beta F405A chain having a C-terminal c-myc tag to generate the final VH(anti-hCD19)-CH13-BT beta c-myc chain. Subsequently, the two chains were ligated independently in the modified pREP4 vector described in example 1. The BT alpha His chain originated from example 1 (SEQ ID NO: 10).

[0680] For transient expression of the V1H(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule, equal quantities of the three engineered chain vectors were co-transfected into suspension-adapted HEK293-EBNA cells as described previously (example 1). The FAB engineered hetero-dimeric construct was then purified from the cell-free supernatant using protein-A affinity chromatography and used for further analysis (procedure according to example 1). The correct assembly of the FAB engineered hetero-dimer can be assessed by SDS-PAGE analysis of samples under non-reducing conditions, since the correctly assembled molecule is expected to have a different SDS-PAGE mobility from the possible homo-dimeric species.

[0681] Transient transfection yields were 50 mg/l. The results of a typical SDS-PAGE analysis are shown in FIG. 17. Post protein-A purification the VH(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule (lane1) is the main species produced (A) with some homo-dimeric species (B) and some half-molecule (C) present.

[0682] It is possible to assess the proportion of hetero-dimer to homo-dimer in the protein-A purified preparation by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands (procedure according to example 1). FIG. 18 shows that the VH(anti-hCD19)-CH13-BT beta c-myc_BT alpha His_VL(anti-hCD19)-CK3 molecule represents at least 51.9% of the protein-A purified material.

[0683] In conclusion, this example shows that the novel FAB fragment with engineered protein-protein interfaces based on the protein-protein interface of the homo-dimeric CH3 (.gamma.1) constant domain is suitable for building further therapeutic hetero-dimeric immunoglobulins; more precisely, immunoglobulin molecules comprising two novel hetero-dimeric pairs of constant domains, one pair having engineered protein-protein interfaces based on the human T-cell receptor constant domain alpha and beta, and the other pair having engineered protein-protein interfaces based on the homo-dimeric CH3 (.gamma.1) constant domain.

Example 7: Immunoglobulin Domains with Engineered Protein-Protein Interfaces Derived from Naturally Occurring Homo-Dimeric or Hetero-Dimeric Immunoglobulin Super-Family Members

[0684] This present example provides various set of mutations in human immunoglobulin domains to create novel protein-protein interfaces in acceptor domains and allow for one engineered domain to interact by hetero-dimerization or homo-dimerization with another engineered domain.

[0685] The mutations can be derived from naturally occurring dimeric immunoglobulin super-family members for example from the naturally occurring homo-dimeric human IGHG1 CH3 domain or from a naturally occurring hetero-dimers such as the human TCR.alpha. (IMGT.RTM. reference TRAC, the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acid Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3): 185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)), TCR.beta. (IMGT.RTM. reference TRBC1 and TRBC2), TCR.gamma. (IMGT.RTM. reference TRGCC1, TRGC2(2.times.), and TRGC2(3.times.)), and TCR.delta. (IMGT.RTM. reference TRDC) constant domains and introduced into the human heavy chain immunoglobulin constant domains of the .gamma.1, .gamma.2, .gamma.3, .gamma.4, .alpha.1, .alpha.2, .epsilon., and .mu. isotypes (IMGT.RTM. reference IGHG1, IGHG2, IGHG3, IGHG4, IGHA1, IGHA2, IGHE, IGHM, respectively) or into the human immunoglobulin light chain constant domain .kappa. (IMGT.RTM. reference IGKC) or into the human immunoglobulin light chain constant domains .kappa. .lamda.2, .lamda.3, .lamda.6, and .lamda.7 (IMGT.RTM. reference IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7, respectively).

[0686] In this example, amino acid substitutions were derived from the analysis of overlays of 3D structures of TCR domains (PDB codes: IKGC or 1J8H (TCR.alpha.-TCR.beta.) and 1HXM (TCR.delta.-TCR.gamma.)) with 3D structures of human IgG domains which were either retrieved form public databases (see Table 2) or modelled depending on availability (CH1 domains of .alpha.1, .alpha.2, .epsilon.; CH2 domains of .gamma.2, .gamma.3, .alpha.2, .mu.; CH3 domains of .gamma.2, .gamma.3, .alpha.2, .mu. and CH4 domain of .mu.). In addition, amino acid substitutions were derived from the analysis of overlays of 3D structures of human IgG domains in a pair wise fashion; for example, the human (IGHG1 CH3 homo-dimeric pair of domains was overlaid onto the human IGHG1 CH1-C.kappa. hetero-dimer. Models were calculated using the structure homology-modelling server SWISS-MODEL (Arnold K et al., Bioinformatics, 22(2): 195-201 (2006); http://swissmodel.expasy.org) in the automated mode with the following inputs: the protein domain amino acid sequence retrieved from the IMGT.RTM. database with specific amino acid substitutions depending on the project requirements (the international ImMunoGeneTics information System.RTM.; Lefranc M P et al, Nucleic Acids Res. 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(t):219-21 (2000); Lefranc M P, Nucleic Acid Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)), and a 3D structure template consisting of an experimentally solved 3D structure from a related protein domain automatically identified by the SWISS-MODEL software. All experimentally solved 3D structures were retrieved from the RCSB Protein Data Bank (www.pdb.org; Berman H M et al., Nucleic Acids Res, 28(1):235-42 (2000)). Overlays were performed using the Coot software (Emsley P and Cowtan K, Acta Crystallogr D Biol Crystallogr, 60(Pt 12 Pt 1):2126-32 (2004)). Visualization for analysis was done in the Discovery-Studio software from Accelrys (Cambridge, UK). The selection of each subset of mutations followed similar criteria to the ones described in example 1.

[0687] Examples of homo-dimeric or hetero-dimeric immunoglobulin domains to be used as donor domains or parent domain are selected from the group consisting of human IGHA1 CH1, IGHA1 CH2, IGHA1 CH3, IGHA2 CH1, IGHA2 CH2, IGHA2 CH3, IGHD CH1, IGHD CH2, IGHD CH3, IGHG1 CH1, IGHG1 CH2, IGHG1 CH3, IGH2 CH1, IGHG2 CH2, IGHG2 CH3, IGHG3 CH1, IGHG3 CH2, IGHG3 CH3, IGHG4 CH1, IGHG4 CH2, IGHG4 CH3, IGHGP CH1, IGHGP CH2, IGHGP CH3, IGHE CH1, IGHE CH2, IGHE CH3, IGHE CH4, IGHM CH1, IGHM CH2, IGHM CH3, IGHM CH4, IGKC, IGLC1, IGLC2, IGLC3, IGLC6, IGLC7, TRAC, TRBC1, TRBC2, TRDC, TRGC1, TRGC2 (2.times.), and TRGC2 (3.times.).

TABLE-US-00002 TABLE 2 PDB codes used for the overlay analysis of human IgG molecules. Domain Isotype CH1 CH2--CH3 CH4 CH.kappa. C.lamda. .gamma.1 1VGE 1H3U 1VGE 7FAB .gamma.2 3KYM .gamma.3 1Q1J .gamma.4 3NAA 1ADQ .mu. 1QLR .alpha.1 1OW0 .epsilon. 2WQR 2WQR

[0688] Details of the amino acid variations for the CH1-CH3 domains between the human IGHG1, IGHG2, IGHG3, and IGHG4 isotypes can be found in FIG. 19.

[0689] Complete lists of the substitutions derived from the protein-protein interface of the human TCR constant domains (TRAC, TRBC2, TRDC, and TRGC1) or the human IGHG1 CH3 domain that can be introduced in the human IGHG1-G4 CH1, IGHG1-G4 CH2, IGHG1-G4 CH3, IHGE CH4, IGHM CH4, IGKC, and IGLC (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7) protein-protein interfaces are provided in FIG. 20-23 which corresponds to IMGT.RTM. numbering as referenced in the IMGT.RTM. database (http://imgt.cines.fr/, the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res. 27(1); 209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefing in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0690] A complete overview of the preferred positions considered for substitution with key amino acid residues derived from the protein-protein interface of the TCR constant domains (.alpha., .beta., .delta., .gamma.) or the CH3 domain of IGHG1 is also provided below. Since the IMGT.RTM. numbering is based on the comparative analysis of the 3D structures of the immunoglobulin super-family domains, it defines the 3D equivalent positions of the substitutions between donor and acceptor (parent) domains.

7.1 Examples of Molecular Modelling.

[0691] 7.1.1 Modelling Hetero-Dimeric Domain Pain Based on the Human TCR.alpha.-TCR.beta. constant domain protein-protein interface.

[0692] a. TCR.alpha. Based Substitutions.

[0693] The amino acid sequence positions from the protein-protein interface of the human TCR.alpha. constant domain (donor domain) which are used to substitute the 3D equivalent amino acid sequence positions from the protein-protein interface of the parent immunoglobulin domain are listed below:

[0694] The donor positions from human TCR.alpha. are as follows: 20, 22, 26, 79, 85.1, 86, 88, and 90 (having the following amino acids: K20, V22, T26, Y79, S85.1, V86, W88, and N90), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering (the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc M P, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3): 185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)),

[0695] The acceptor positions in all human immunoglobulin domains are as follows: 20, 22, 26, 79, 85.1, 86, 88, and 90; except for position 79 in .alpha.1: CH2 (78), .alpha.2: CH2 (78), .epsilon.: CH2 (81) and .epsilon.: CH3 (80), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0696] b. TCR Based Substitutions.

[0697] The amino acid sequence positions from the protein-protein interface of the human TCR.beta. constant domain (donor domain) which are used to substitute the 3D equivalent amino acid sequence positions from the protein-protein interface of the parent immunoglobulin domain are listed below:

[0698] The donor positions from the human TCR beta are as follows: 3, 5, 7, 20, 22, 26, 81, 84.1, 84.3, 85.1, 86, 88, and 90 (having the following amino acids: E3, A5, F7, T20, V22, T26, D81, L84.1, E84.3, C85.1 or A85.1 or S85.1, S86, R88, and R90), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0699] The acceptor positions in all human immunoglobulin domains are as follows: 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 85.1, 86, 88, and 90; except for position 81 in .alpha.1: CH2 (80), .alpha.2: CH2 (80), .epsilon.: CH2 (83); except for position 84 in .alpha.1: CH2 (83), .alpha.2: CH2 (83); except for position 84.2 in .alpha.1: CH2 (84), .alpha.2: CH2 (84), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0700] c. Modelling the Human TCR.alpha.-TCR.beta. Constant Domain Protein-Protein Interface on the Human IGHG1 CH3 Homo-Dimer.

[0701] To model a novel hetero-dimer based on the human IGHG1 CH3 homo-dimer with protein-protein interfaces derived from the human TCR.alpha./.beta. constant domains, two models of a human IGHG1 CH3 monomer substituted in its protein-protein interface with selected amino acids were calculated (see above for methods). One monomer was substituted at selected equivalent 3D positions in its protein-protein interface with amino acids from the protein-protein interface of the human TCR.alpha. constant domain, and a second monomer was substituted at selected equivalent 3D positions in its protein-protein interface with amino acids from the protein-protein interface of the human TCR.beta. constant domain. The Ca traces of the resulting models for the two engineered protein domains were further overlaid on the experimentally solved parental CH3 homo-dimer 3D structure (PDB code 1H3U) for visual inspection. In addition, a computational analysis of the protein-protein interface of the engineered domains was performed using the online PIC web server (http://crick.mbu.iisc.cmet.in/.about.PIC/, Tina K G et al., Nucleic Acids Res. 35(Web Server issue):W473-6 (2007)). Tables 3 and 4 list the interacting residues in the protein-protein interface of the engineered hetero-dimer identified from computational analysis, and direct measurements; the amino acid position of each group member is indicated according to the IMGT.RTM. numbering. From the modelling of a large number of immunoglobulin domain pairs engineered in their protein-protein interface with substitutions derived from naturally occurring homo-dimeric and hetero-dimeric protein-protein interface residues, the residues 20, 22, 26, 85.1, 86 and 88, in particular the residues 22, 85.1, 86 and 88 (numbering according to IMGT.RTM.) were repeatedly found to provide the most important contacts between the protein-protein interfaces of the engineered domains. More generally, by visual inspection and calculation of protein-protein interactions (using the online PIC web server (http://crick.mbu.iisc.emet.in/.about.PIC/, Tina K G et al., Nucleic Acids Res, 35(Web Server issue); W473-6 (2007) or other protein-protein interaction programs known in the art), the residues 20, 22, 26, 85.1, 86 and 88, in particular the residues 22, 85.1, 86 and 88 were found to mediate the most important interactions for the hetero-dimerization or homo-dimerization of all engineered domains.

[0702] Without being bound by theory, the analysis of the newly engineered domain protein-protein interfaces identified four important subsets of interacting residues: residue 88 in the first engineered immunoglobulin (BT alpha) interacts with residue 85.1 and 86 in the second engineered immunoglobulin (BT beta), residue 85.1 in the first engineered immunoglobulin (BT alpha) interacts with residue 86 in the second engineered immunoglobulin (BT beta), residue 22 in the first engineered immunoglobulin (BT alpha) interacts with residue 22 and residue 86 in the second engineered immunoglobulin (BT beta), and residue 20 in the first engineered immunoglobulin (BT alpha) interacts with residue 26 in the second engineered immunoglobulin (BT beta). Conversely, residue 88 in the second engineered immunoglobulin (BT beta) interacts with residue 85.1 and 86 in the first engineered immunoglobulin (BT alpha), residue 85.1 in the second engineered immunoglobulin (BT beta) interacts with residue 86 in the first engineered immunoglobulin (BT alpha), residue 22 in the second engineered immunoglobulin (BT beta) interacts with residue 22 and residue 86 in the first engineered immunoglobulin (lit alpha), and residue 20 in the second engineered immunoglobulin (BT beta) interacts with residue 26 in the first engineered immunoglobulin (BT alpha).

TABLE-US-00003 TABLE 3 Interactions between positions from the protein-protein interface of the TCR.alpha.-CH3 monomer to the protein-protein interface of TCR.beta.-CH3 monomer in the newly engineered CH3 hetero-dimer. The IMGT .RTM. numbering is used. TCR.alpha.-CH3 TCR.beta.-CH3 (BT alpha) (BT beta) 20 3, 5, 7, 26, 84.2, 85.1, 86 22 5, 7, 22, 86, 88 26 20, 88, 90 79 26, 81, 84, 85.1, 86 85.1 20, 81, 84, 86, 88 86 22, 81, 84, 86, 88 88 26, 81, 84, 84.2, 85.1, 86 90 26, 84.2, 85.1

TABLE-US-00004 TABLE 4 Interactions between positions from the protein-protein interface of the TCR.beta.-CH3 monomer to the protein-protein interface of TCR.alpha.-CH3 monomer in the newly engineered CH3 hetero-dimer. The IMGT .RTM. numbering is used. TCR.beta.-CH3 TCR.alpha.-CH3 (BT beta) (BT alpha) 3 20 5 20, 22 7 20, 22 20 26, 85.1, 86 22 22, 86 26 20, 79, 88, 90 81 79, 85.1, 86, 88 84 79, 86, 88 84.2 20, 79, 88, 90 85.1 20, 79, 86, 88, 90 86 20, 22, 79, 85.1, 86 88 22, 26, 85.1, 86 90 26, 85.1

7.1.2 Modelling Hetero-Dimeric Domain Pairs Based on the Human TCR.delta.-TCR.gamma. Constant Domain Protein-Protein Interface

[0703] a. TCR.delta. Based Substitutions.

[0704] The amino acid sequence positions from the protein-protein interface of the human TCR.delta. constant domain (donor domain) which are used to substitute the 3D equivalent amino acid sequence positions from the protein-protein interface of the parent immunoglobulin domain are listed below:

[0705] The donor positions from the human TCR delta are as follows: 7, 20, 22, 27, 79, 82, 84.2, 85.1, 86, 88, and 90 (having the following amino acids: M7, N20, A22, E27, F79, A82, S84.2, N85.1, V86, L88, and K90), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0706] The acceptor positions in all human immunoglobulin domains are as follows: 7, 20, 22, 27, 79, 81, 84.2, 85.1, 86, 88, 90; except for position 79 in .alpha.1: CH2 (78), .alpha.2: CH2 (78), .epsilon.: CH2 (81) and .epsilon.; CH3 (80); except for position 81 in .alpha.1: CH2 (80), .alpha.2: CH2 (80), .epsilon.: CH2 (83) and .epsilon.: CH3 (82); except for position 84.2 in .alpha.1: CH2 (84), .alpha.2: CH2 (84), .epsilon.: CH2 (84.4), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0707] b. TCR.gamma. Based Substitutions.

[0708] The amino acid sequence positions from the protein-protein interface of the human TCR.gamma. constant domain (donor domain) which are used to substitute the 3D equivalent amino acid sequence positions from the protein-protein interface of the parent immunoglobulin domain are listed below:

[0709] The door positions from the human TCR gamma are as follows: 3, 5, 20, 22, 26, 27, 81, 84, 85.1, 86, and 88 (having the following amino acids: K3, T5, T20, L22, E26, K27, G81, M84, M85.1, F86, and W88), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0710] The acceptor positions in all human immunoglobulin domains are as follows: 3, 5, 20, 22, 26, 27, 81, 84, 85.1, 86, 88; except for position 81 in .alpha.1: CH2 (80), .alpha.2: CH2 (80), .epsilon.: CH2 (83); except for position 84 in .gamma.1, .gamma.2, .gamma.3, .gamma.3: CH2 (for all 83), .alpha.1: CH2 (83), .alpha.2: CH2 (83), .epsilon.: CH2 (84.1), .mu.: CH2 (83) and .mu.: CH3 (83), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0711] c. Modelling the Human TCR.delta.-TCR.gamma. Constant Domain Protein-Protein Interface on the Human IGHG1 CH3 Homo-Dimer.

[0712] To model a novel hetero-dimer based on the human IGHG1 CH3 homo-dimer with protein-protein interfaces derived from the human TCR.delta./.gamma. constant domains, two models of a human IGHG1 CH3 monomer substituted in its protein-protein interface with selected amino acids were calculated (see above for methods). One monomer was substituted at selected equivalent 3D positions in its protein-protein interface with amino acids from the protein-protein interface of the human TCR.delta. constant domain, and a second monomer was substituted at selected equivalent 3D positions in its protein-protein interface with amino acids from the protein-protein interface of the human TCR.gamma. constant domain. The C.alpha. traces of the resulting models for the two engineered protein domains were further overlaid on the experimentally solved parental CH3 homo-dimer 3D structure (PDB code 1H3U) for visual inspection. In addition, a computational analysis of the protein-protein interface of the engineered domains was performed using the online PIC web server (http://crick.mbu.iisc.ernet.in/.about.PIC, Tina K G et al., Nucleic Acids Res, 35(Web Server issue):W473-6 (2007)). Tables 5 and 6 list the interacting residues in the protein-protein interface of the engineered hetero-dimer identified from computational analysis, and direct measurements; the amino acid position of each group member is indicated according to the IMGT.RTM. numbering. From the modelling of a large number of immunoglobulin domain pairs engineered in their protein-protein interface with substitutions derived from naturally occurring homo-dimeric and hetero-dimeric protein-protein interface residues, the residues 20, 22, 26, 85.1, 86 and 88, in particular the residues 22, 85.1, 86 and 88 (numbering according to IMGT.RTM.) were repeatedly found to provide the most important contacts between the protein-protein interfaces of the engineered domains. More generally, by visual inspection and calculation of protein-protein interactions (using the online PIC web server (http://crick.mbu.iisc.emet.in/.about.PIC/, Tina K G et al., Nucleic Acid Res, 35(Web Server issue):W473-6 (2007) or other protein-protein interaction programs known in the art), the residues 20, 22, 26, 85.1, 86 and 88, in particular the residues 22, 85.1, 86 and 88 were found to mediate the most important interactions for the hetero-dimerization or homo-dimerization of all engineered domains.

[0713] Without being bound by theory, the analysis of the newly engineered domain protein-protein interfaces identified four important subsets of interacting residues: residue 88 in the first engineered immunoglobulin (BT delta) interacts with residue 85.1 and 86 in the second engineered immunoglobulin (BT gamma), residue 85.1 in the first engineered immunoglobulin (BT delta) interacts with residue 86 in the second engineered immunoglobulin (BT gamma), residue 22 in the first engineered immunoglobulin (BT delta) interacts with residue 22 and residue 86 in the second engineered immunoglobulin (BT gamma), and residue 20 in the first engineered immunoglobulin (BT delta) interacts with residue 26 in the second engineered immunoglobulin (BT gamma). Conversely, residue 88 in the second engineered immunoglobulin (BT gamma) interacts with residue 85.1 and 86 in the first engineered immunoglobulin (BT delta), residue 85.1 in the second engineered immunoglobulin (BT gamma) interacts with residue 86 in the first engineered immunoglobulin (BT delta), residue 22 in the second engineered immunoglobulin (BT gamma) interacts with residue 22 and residue 86 in the first engineered immunoglobulin (BT delta), and residue 20 in the second engineered immunoglobulin (UT gamma) interacts with residue 26 in the first engineered immunoglobulin (BT delta).

TABLE-US-00005 TABLE 5 Interactions between positions from the protein-protein interface of the TCR.delta.-CH3 monomer to the protein-protein interface of TCR.gamma.-CH3 monomer in the newly engineered CH3 hetero-dimer. The IMGT .RTM. numbering is used. TCR.delta.-CH3 TCR.gamma.-CH3 (BT delta) (BT gamma) 7 20, 22 20 3, 5, 26, 27, 85.1, 86 22 5, 22, 26, 86 26 20, 88 27 13 79 26, 81, 84, 85.1, 86 81 81, 84, 85.1, 86 84.2 20, 81, 88 85.1 20, 81, 86, 88 86 20, 22, 84, 86, 88 88 26, 81, 84, 85.1, 86 90 26, 27, 85.1, 90

TABLE-US-00006 TABLE 6 Interactions between positions from the protein-protein interface of the TCR.gamma.-CH3 monomer to the protein-protein interface of TCR.delta.-CH3 monomer in the newly engineered CH3 hetero-dimer. The IMGT .RTM. numbering is used. TCR.gamma.-CH3 TCR.gamma.-CH3 (BT gamma) (BT delta) 3 20 5 20, 22 20 7, 26, 84.2, 85.1, 86 22 7, 22, 79, 86 26 20, 79, 88, 90 27 20, 90 81 81, 84.2, 85.1, 88 84 79, 81, 86, 88 85.1 20, 79, 81, 86, 88, 90 86 20, 22, 79, 81, 85.1, 86, 88 88 84.2, 85.1, 86

7.13 Modelling Hetero-Dimeric Domain Pairs Based on the Human IGHG1 CH3 Constant Domain Pair Protein-Protein Interface.

[0714] The amino acid sequence positions from the protein-protein interface of the human IGHG1 CH3 constant domain (donor domain) which are used to substitute the 3D equivalent amino acid sequence positions from the protein-protein interface of the parent immunoglobulin domain are listed below:

[0715] a. A first set of donor positions from the human CH3 of IGHG1 comprises the following positions: 3, 5, 12, 13, 18, 20, 22, 79, 81, 84.2, 85.1, 86, and 88 (having the following amino acids: Q3, Y5, D12, E13, Q18, S20, T22, K79, T81, D84.2, F85.1, Y86, and K88), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0716] The acceptor positions for the first set in all human immunoglobulin domains except for all human immunoglobulin light chain domains are as follows: 3, 5, 12, 13, 18, 20, 22, 79, 81, 84.2, 85.1, 86, and 88, except for position 81 in .gamma.1, .gamma.2, .gamma.3, .gamma.3: CH2 (for all 83), except for position 84.2 in .gamma.1, .gamma.2, .gamma.3, .gamma.3: CH2 (for all 84.3), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0717] b. A second set of donor positions from the human CH3 of IGHG1 comprises the following positions: 3, 5, 7, 12, 13, 22, 26, 79, 81, 84, 85.1, 86, and 88 (having the following amino acids; Q3, Y5, L7, D12, E13, T22, K26, K79, T81, V84, F85.1, Y86, and K88), wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0718] The acceptor positions for the second set in all human immunoglobulin light chain domains are as follows: 3, 5, 7, 12, 13, 22, 26, 79, 81, 83, 85.1, 86, and 88, except for a human IGLC domain where the second set in is as follows: 3, 5, 7, 11, 12, 22, 26, 79, 81, 83, 85.1, 86, and 88, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

[0719] c. Modelling the Human IGHG1 CH3 Homo-Dimer Protein-Protein Interface on the Human CH1-CK Hetero-Dimer Protein-Protein Interface.

[0720] A model for the human IGHG1 CH1 domain substituted in its protein-protein interface with amino acids at equivalent 3D positions derived from the protein-protein interface of a human IGHG1 CH3 constant domain (CH13), and a model for the human C.kappa. domain substituted in its protein-protein interface with amino acids at equivalent 3D positions derived from the protein-protein interface of a human IGHG1 CH3 constant domain (CK3) were calculated (see above for methods). The Ca traces of the resulting models for the two engineered protein domains were further overlaid on the experimentally solved parental human IGHG1 CH1-C.kappa. hetero-dimer 3D structure (PDB code 1VGE) for visual inspection. In addition, a computational analysis of the protein-protein interface of the engineered domains was performed using the online PIC web server (http://crick.mbu.iisc.ernet.in/.about.PIC/, Tina K G et al., Nucleic Acids Res, 35(Web Server issue):W473-6 (2007)). Tables 7 and 8 list the interacting residues in the protein-protein interface of the engineered hetero-dimer identified from computational analysis, and direct measurements; the amino acid position of each group member is indicated according to the IMGT.RTM. numbering. From the modelling of a large number of immunoglobulin domain pairs engineered in their protein-protein interface with substitutions derived from naturally occurring homo-dimeric and hetero-dimeric protein-protein interface residues, the residues 20, 22, 26, 85.1, 86 and 88, in particular the residues 22, 85.1, 86 and 88 (numbering according to IMGT.RTM.) were repeatedly found to provide the most important contacts between the protein-protein interfaces of the engineered domains. More generally, by visual inspection and calculation of protein-protein interactions (using the online PIC web server (http://crick.mbu.iisc.ernet.in/.about.PIC/, Tina K G et al., Nucleic Acids Res, 35(Web Server issue):W473-6 (2007) or other protein-protein interaction programs known in the art), the residues 20, 22, 26, 85.1, 86 and 88, in particular the residues 22, 85.1, 86 and 88 were found to mediate the most important interactions for the hetero-dimerization or homo-dimerization of all engineered domains.

[0721] Without being bound by theory, the analysis of the newly engineered domain protein-protein interfaces identified four important subsets of interacting residues: residue 88 in the first engineered immunoglobulin (CH13) interacts with residue 85.1 and 86 in the second engineered immunoglobulin (CK3), residue 85.1 in the first engineered immunoglobulin (CH13) interacts with residue 86 in the second engineered immunoglobulin (CK3), residue 22 in the first engineered immunoglobulin (CH13) interacts with residue 22 and residue 86 in the second engineered immunoglobulin (CK3), and residue 20 in the first engineered immunoglobulin (CH13) interacts with residue 26 in the second engineered immunoglobulin (CK3). Conversely, residue 88 in the second engineered immunoglobulin (CK3) interacts with residue 85.1 and 86 in the first engineered immunoglobulin (CH13), residue 85.1 in the second engineered immunoglobulin (CK3) interacts with residue 86 in the first engineered immunoglobulin (CH13), residue 22 in the second engineered immunoglobulin (CK3) interacts with residue 22 and residue 86 in the first engineered immunoglobulin (CH13), and residue 20 in the second engineered immunoglobulin (CK3) interacts with residue 26 in the first engineered immunoglobulin (CH13),

TABLE-US-00007 TABLE 7 Interactions between positions from the protein-protein interface of the CH13 monomer to the protein-protein interface of CK3 monomer in the newly engineered CH1-CK hetero-dimer. The IMGT .RTM. numbering is used. CH13 CK3 3 13 5 12, 13 12 79, 88 13 3, 5, 7 18 3, 5, 26 20 3, 5, 7, 26, 85.1, 86 22 7, 22, 86 26 20 79 26, 81, 83, 85.1, 86 81 79, 81, 83, 85.1, 86 84.2 79, 88 85.1 79, 81, 86, 88 86 79, 81, 83, 85.1, 86 88 26, 81, 83, 85.1, 86

TABLE-US-00008 TABLE 8 Interactions between positions from the protein-protein interface of the CK3 monomer to the protein-protein interface of CH13 monomer in the newly engineered CH1-CK hetero-dimer. The IMGT .RTM. numbering is used. CK3 CH13 3 13 5 12, 13 12 79, 88 13 3, 5, 7 18 3, 5, 26 20 3, 5, 7, 26, 85.1, 86 22 7, 22, 86 26 20 79 26, 81, 83, 85.1, 86 81 79, 81, 83, 85.1, 86 84.2 79, 88 85.1 79, 81, 86, 88 86 79, 81, 83, 85.1, 86 88 26, 81, 83, 85.1, 86

7.2 Summary of Donor Positions Derived from Human TCR Constant Domains (TRAC, TRBC2, TRDC, TRGC1) and IGHG1 CH3.

[0722] A summary of the donor positions derived from the human TRAC (TCR.alpha., abbreviated ALPHA), TRBC2 (TCR.beta., abbreviated BETA), TRDC (TCR.delta., abbreviated DELTA), TRGC1 (TCR.gamma., abbreviated GAMMA) and IGHG1 CH3 (abbreviated CH43) is found in Table 9. The amino acid position of each group member is indicated according to the IMGT.RTM. numbering. Amino acid positions for CH3 (set i) are donor positions for substituting all human immunoglobulin domains except for light chain domains. Amino acid positions for CH3 (set ii) are donor positions for substituting all human immunoglobulin light chain domains.

TABLE-US-00009 TABLE 9 Donor position derived from the human TRAC, TRBC2, TRDC, TRGC1 and IGHG1 CH3. The IMGT .RTM. numbering is used. IMGT .RTM. 3 5 7 12 13 18 20 22 26 27 79 ALPHA K V T Y BETA E A F T V T DELTA M N A E F GAMMA K T T L E K CH3 set i Q V D E Q S T K CH3 set ii Q Y L D E T K K IMGT .RTM. 81 82 84 84.1 84.2 84.3 85.1 86 88 90 ALPHA S V W N BETA D L E C* S R R DELTA A S N V L K GAMMA G M M F W CH3set i T D F Y K CH3set ii T V F Y K (*or C or S)

7.3 Basis for Additional Substitutions.

[0723] In a more general approach, the protein-protein interface residues from naturally occurring dimeric protein domains of the immunoglobulin super-family selected from the group consisting of 3, 5, 7, 12, 13, 18, 20, 22, 26, 27, 79, 81, 82, 84, 84.1, 84.2, 84.3, 85.1, 86, 88, and 90, can be used as donor residues to create new dimers, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering. Table 10 shows the amino acid residues of selected positions 20, 22, 26, 85.1, 86, and 88 from the hetero-dimeric donor domain CH1, and homo-dimeric domains CH2, CH3 and CH4 originating from different isotypes which can be used to substitute amino acids at the same positions in parent domains. Table 11 shows the amino acid residues of selected positions 20, 22, 26, 85.1, 86, and 88 from various hetero-dimeric human immunoglobulin superfamily members which can be used to substitute amino acids at the same positions in parent domains.

TABLE-US-00010 TABLE 10 Amino acid residues of selected positions 20, 22, 26, 85.1, 86, and 88 of the hetero-dimeric donor domain CH1, and homo-dimeric CH2, CH3 and CH4 domains. The IMGT .RTM. numbering is used. 20 22 26 85.1 86 88 CH1 IGHA1 V A Q T S Q IGHA2 V A Q T S Q IGHD V A T M S Q IGHE T G T A I L IGHG1 A G K S S V IGHG2 A G K S S V IGHG3 A G K S S V IGHG4 A G K S S V IGHGP A G K S S V IGHM A G Q A T Q CH2 IGHA1 N T T S S V IGHA2 N T T S S V IGHD T T V S H R IGHE Q L S S Q E IGHG1 E T V R V V IGHG2 E T V R V V IGHG3 E T V R V V IGHG4 E T V R V V IGHGP E T V H V V IGHM K I T K T T CH3 IGHA1 T T R A T I IGHA2 T T R A T I IGHD W L S W W V IGHE T T V T T T IGHG1 S T K F Y K IGHG2 S T K F Y K IGHG3 S T K F Y K IGHG4 S T K F Y R IGHGP T T K F Y K IGHM K T T S V E CH4 IGHE T A Q F F R IGHM T T T F H I

TABLE-US-00011 TABLE 11 Amino acid residues of selected positions 20, 22, 26, 85.1, 86, and 88 of various hetero-dimeric human immunoglobulin superfamily members. The IMGT .RTM. numbering is used. 20 22 26 85.1 86 88 Human IGKC S V N S S T Human IGLC1- T V S A S Y IGLC7 Human TRAC K V T S V W Human TRBC1, T V T C S R TRBC2 Human TRDC N A K N V L Human TRGC1, T L E M F W TRGC2 (2x, 3x)

7.4 Correspondence Between the IMGT.RTM. Unique Numbering, the EU Numbering, and the Kabat Numbering.

[0724] The correspondence between the IMGT.RTM. unique numbering and the EU numbering for most of the human heavy chain constant domains cited above is found in Table 12. The correspondence between the IMGT.RTM. unique numbering and the EU numbering for the human kappa constant light chain domain, and the correspondence between the IMGT.RTM. unique numbering and the Kabat numbering for the human lambda constant light chain domains are both found in Table 13.

[0725] A complete correspondence for the human IGHG1 CH1-CH3, IGKC, and IGLC domains is found in FIGS. 19A, 19B, 19C, 19D, 22, 23A, and 23B and alternatively at the IMGT.RTM. database (the international ImMunoGeneTics information System.RTM.; Lefranc M P et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al, Nucleic Acids Res, 28(1):219-21 (2000); Lefranc M P, Nucleic Acids Res, 29(t):207-9 (2001); Lefranc M P, Nucleic Acids Res. 31(1):307-10 (2003); Lefranc M P et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)), more specifically from the pages found at:

[0726] http://imgt.cines.fr/textes/IMGTScientificChart/Numbering/Hu_IGHGnb- er.html;

[0727] http://imgt.cines.fr/textes/IMGTScientificChart/Numbering/Hu_IGKCnb- er.html;

[0728] http://imgt.cines.fr/textes/IMGrTScientificChart/Numbering/Hu_IGLCn- ber.html.

TABLE-US-00012 TABLE 12 Correspondence between the IMGT .RTM. unique numbering and the EU numbering for the CH1, CH2, and CH3 domains of human IGHG1, IGHG2, IGHG3, and IGHG4. IMGT .RTM. 3 5 7 12 13 18 20 22 26 27 79 CH1 124 126 128 133 134 139 141 143 147 148 168 EU CH2 239 241 243 248 249 256 258 260 264 265 288 EU CH3 347 349 351 356 357 362 364 366 370 371 392 EU IMGT .RTM. 81 83 84 84.2 85.1 86 88 90 CH1 170 172 173 175 181 183 185 187 EU CH2 290 292 293 295 301 303 305 307 EU CH3 394 396 397 399 405 407 409 411 EU

TABLE-US-00013 TABLE 13 Correspondence between the IMGT .RTM. unique numbering and the EU numbering for human IGKC, and correspondence between the IMGT .RTM. unique numbering and the Kabat numbering for human IGLC (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7 domains). IMGT .RTM. 3 5 7 12 13 18 20 22 26 27 79 IGKC 114 116 118 123 124 129 131 133 137 138 160 EU IGLC 114 116 118 123 124 129 131 133 137 138 160 Kabat IMGT .RTM. 81 83 84 84.2 85.1 86 88 90 IGKC 162 164 165 167 174 176 178 180 EU IGLC 162 164 165 167 174 176 178 180 Kabat

Example 8: Construction of a Bispecific Hetero-Dimeric Immunoglobulin Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains

[0729] The HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival (Earp H S et al., Breast Cancer Res Treat, 35(1):115-32 (1995)); the receptor family includes four distinct members: epidermal growth factor receptor EGFR (ErbB1, or HER1), HER2 (ErbB2 or p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2) which are generally found in various combinations in cells (Rowinsky E K, Annu Rev Med, 55:433-57 (2004); Hynes N E and Lane H A, Nat Rev Cancer, 5(5):341-54 (2005)). Receptor dimerization is an essential requirement for ErbB function and for the signaling activity of these receptors (Biaselga J and Swain S M, Nat Rev Cancer, 9(7):463-75 (2009)); more specifically, receptor hetero-dimerization is thought to increase the diversity of cellular responses to various HER ligands. Therapeutics that target a single receptor from the HER family are presently in use in treating diseases such as breast cancer, non-small cell lung cancer, colorectal cancer, head and neck cancer and pancreatic cancer (Engelman J A and Janne P A, Clin Cancer Res, 14(10); 2895-9 (2008); Baselga J and Swain S M, Nat Rev Cancer, 9(7):463-75 (2009)). Although these therapies had some success, issues related to native and induced resistance and toxicity are currently limiting their efficacies (Robert C et al., Lancet Oncol, 6(7):491-500 (2005); Jones K L and Buzdar A U, Lancet Oncol, 10(12):1179-87 (2009)). Therapeutics that could target multiple receptors from the HER family may offer improved clinical efficacy and lower toxicity.

[0730] To produce a bispecific molecule that could specifically bind to at least two HER receptors (FIG. 24), the antigen binding sites derived from the recombinant humanized anti-HER2 antibody 4D5 (rhuMAbHER2, huMAB4D5-8, Trastuzumab or Herceptin.RTM.; U.S. Pat. No. 5,821,337), and the recombinant chimeric anti-EGFR antibody C225 (IMC-C225, Cetuximab or Erbitux.RTM.; PCT Publication NO: WO 96/40210) were selected as inputs for gene synthesis at GENEART AG (Regensburg, Germany). For both antibodies, the heavy chain variable sequence (VH) and the light chain variable sequence (VL) were synthesized and reformatted into a scFv fragment (VH and VL domains were fused with a 15 amino acid GlySer linker: (Gly.sub.4Ser).sub.3) and a chimeric FAB fragment (i.e., a murine VH-human CH1 (.gamma.1) chain assembled with a murine VL-human CK chain) for the anti-HER2 antibody 4D5 and the anti-EGFR antibody C225, respectively. All DNA manipulations followed standard molecular biology techniques and primarily involved PCR amplification, and PCR fragment assembly methods. To create the anti-EGFR portion of the bispecific molecule, the anti-EGFR antibody C225 murine VH-human CH1 (.gamma.1) chain was fused upstream of a BT alpha chain described in example 1 (abbreviated VH(anti-EGFR)-CH1-BT alpha chain. SEQ ID NO: 56), while the anti-EGFR antibody C225 murine VL was fused upstream of a human Kappa constant domain (abbreviated VL(anti-EGFR)-CK chain, SEQ ID NO: 57). To create the anti-HER2 portion of the bispecific molecule, the anti-HER2 scFv was fused upstream of a BT beta F405A chain described in example 2 with a short GlySer amino acid linker (GGGS) between the two sequences (abbreviated scFv(anti-HER2)-BT beta chain, SEQ ID NO: 58). All three resulting chains were further ligated independently into the modified pREP4 vector mentioned in example 1 and co-transfected into HEK293-EBNA cells as described in example 1. Protein production was also according to the procedure described in example 1.

[0731] The expected bispecific molecule consisting of one VH(anti-EGFR)-CH1-BT alpha chain, one VL(anti-EGFR)-CK chain, and one scFv(anti-HER2)-BT beta chain (SEQ ID NOs: 59, 60, and 61), is designated "Erbitux FAB-BT alpha_Herceptin scFv-BT beta".

[0732] To isolate the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer from homo-dimers, ion-exchange chromatography (IEX) steps were performed post protein-A purification (procedure according to example 1). First, an anion-IEX step was performed: the protein-A eluate was buffer exchanged (into 50 mM Tris-HCl pH 9.0) and loaded on a 4 ml SourceQ 30 packed column operated on an .ANG.KTA Purifier (both from GE Healthcare Europe GmbH, Glattbrugg, Switzerland). The Erbitux FAB-BT alpha_Herceptin scFv-BT beta was then eluted with a shallow pH gradient ranging from pH 9.0 to pH 5.0 (final buffer condition was 50 mM sodium acetate pH 5.0). Following SDS-PAGE analysis, the fractions containing the majority of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer were pooled and rebuffered to 50 mM sodium acetate pH 5.0. In a second step, cation IEX was performed by loading the rebuffered fractions from the anion IEX step on a 1 ml MonoS column (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) and running a shallow gradient from 50 mM sodium acetate pH 5.0 to 50 mM Tris-HCl pH 9.0. Fractions containing the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer were selected by SDS-PAGE analysis. Finally the purified the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer was polished on a size-exclusion chromatography column (Superdex 200 HR 16/60, GE Healthcare Europe GmbH, Glattbrugg, Switzerland) to remove traces of aggregates and buffer exchange the final bispecific molecule into PBS. FIG. 25 shows a polyacrylamide SDS gel profile after each purification step as well as two control molecules: a scFv-Fc fusion protein and a monoclonal antibody. The production yields from transient transfections were up to 30 mg/l with a hetero-dimerization rate of 49.5% as judged by scanning densitometry analysis of non-reduced SDS-polyacrylamide (4-12%) gel bands (see example 1 for methods) (FIG. 26).

[0733] To assess the stability of Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer, the purified material was analysed using differential scanning calorimetry (DSC). The calorimetric measurements were carried out on a VP-DSC differential scanning microcalorimeter (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) as described in example 2. The cell volume was 0.128 ml, the heating rate was 1.degree. C./min, and the excess pressure was kept at 64 p.s.i. The protein was used at a concentration of 0.95 mg/ml in PBS (pH 7.4). FIG. 27 shows a thermogram of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer exhibiting two thermal transitions at 69.5 and 75.9.degree. C. It is important to note that no sharp decrease after the heat absorption peak was recorded, indicating that no precipitation or aggregate formation occurred after thermal unfolding (Liu H et al., Immunol Lett, 106(2): 144-53 (2006)). Hence, the melting transitions observed for the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer are similar to the ones observed for a human IGHG4 Fc fragment CH2 and CH3 domains, and similar to the melting transitions generally observed for stable human FAB fragments (Garber E and Demarest S J, Biochem Biophys Res Commun, 355(3):751-7 (2007)). In conclusion, this thermal unfolding study shows that the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer with engineered protein-protein interface based on the TCR alpha and beta constant domains has similar thermo-stability to naturally occurring immunoglobulins.

[0734] The ability of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta heterodimer to bind simultaneously to both of its cognate antigens (human EGFR and human HER2) was tested by surface plasmon resonance (SPR) on a BIAcore 2000 instrument (GE Healthcare Europe GmbH, Glattbrugg, Switzerland). In the present example, the recombinant extracellular domains from the human EGFR and HER2 receptors were individually fused upstream of a human Fc (.gamma.1) fragment (abbreviated EGFR-Fc and HER2-Fc, SEQ ID NO: 62 and 63, respectively). Briefly, the extracellular domains of the human EGFR and HER2 were amplified by PCR from the imaGenes clone NO: EX-A0275-MO2 and Ex-BOO17-M10 (imaGenes GmbH, campus Berlin-Buch, Berlin, Germany), respectively. PCR products were then fused upstream of a human Fc (.gamma.1) portion using PCR assembly methods (SEQ ID NO: 64 and 65, respectively) and independently cloned into a mammalian cell expression vector based on the pcDNA3.1 vector DNA from Invitrogen (Invitrogen AG, Basel, Switzerland). Finally each recombinant vector was transfected into HEK293 cells; after 4-5 days post transfection, supernatants were harvested and recombinant proteins were purified to homogeneity by protein-A affinity chromatography. SPR experiments were performed at 25.degree. C. in 1.times.HBS-EP buffer (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) at a flow rate of 30 .mu.l/min. The purified Fc-fused antigens were immobilized on a research-grade CMS chip (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) via amine coupling using a standard protocol provided by the manufacturer. EFGR-Fc and HER2-Fc, which were immobilized on different channels of the chip led to a signal of 410 and 880 response units (RU), respectively. A channel lacking immobilized antigens was used as reference channel. Responses from this channel were subtracted from measurements. For the binding and co-binding experiments, the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer and the EGFR-Fc were diluted in 1.times.HBS-EP buffer (Healthcare Europe GmbH, Glattbrugg, Switzerland) to a final concentration of 25 and 100 nM, respectively. For all analytes the injections were limited to two minutes. FIG. 28A shows the binding and dissociation between the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer and the immobilized EGFR-Fc. Co-binding of the Erbitux FAB-BT alpha_Herceptin scFv-BT beta and the EGFR-Fc as analytes to the immobilized HER2-Fc is shown in FIG. 28B. In this binding experiment, the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer was first injected on the channel with immobilized HER2-Fc, showing the binding of Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer to the antigen. Three minutes after this first injection, EGFR-Fe was injected, resulting in a co-binding event with the Erbitux FAB-BT alpha_Herceptin scFv-BT beta hetero-dimer, to verify that the observed response was a true co-binding event and not an interaction between both antigens, the EGFR-Fc was injected on the immobilized HER2-Fc and no significant response was recorded (FIG. 28B).

Example 9: Construction of a Bispecific Hetero-Dimeric Immunoglobulin Having a CH3-CH3 Protein-Protein Interface Based on the Human .alpha./.beta. T Cell Receptor Constant Domains, and a FAB Fragment Having a CH1-C.kappa. Protein-Protein Interface Based on the Hetero-Dimeric .delta./.gamma. T Cell Receptor Constant Domains

[0735] Example 5 provides a novel set of modified CH1 (.gamma.1) and C.kappa. constant domains based on the protein-protein interfaces of the human TCR constant domains delta and gamma. This new pair of FAB constant domains offers the opportunity to create human bispecific antibodies without having to compromise in using a common light chain.

[0736] This novel type of bispecific antibody will be based on two distinct heavy chains having different variable domains, and two distinct light chains having different variable domains. The correct assembly of the four distinct chains of the bispecific antibody is based on two principles: (1) to force the two different antibody heavy chains to hetero-dimerize, the novel pair of engineered hetero-dimeric CH3 domains described in example 1-3 will be used, (2) to circumvent any potential light chain mispairing, the novel set of CH1 (.gamma.1) and C.kappa. constant domains described in example 5 will be introduced in one of the two FAB arms of the bispecific antibody. Hence, the bispecific antibody will encompass at least three different protein-protein interfaces in three different portions: the first FAB arm having the naturally occurring hetero-dimeric pair of CH1 (.gamma.1) and C.kappa. constant domains, the second FAB arm having the novel hetero-dimeric pair of CH1 (.gamma.1) and C.kappa. constant domains with protein-protein interfaces originating from the hetero-dimeric pair of TCR delta and TCR gamma constant domains, and one Fc fragment having the novel hetero-dimeric pair of engineered CH3 (.gamma.1) constant domains with protein-protein interfaces originating from the hetero-dimeric TCR alpha and TCR beta constant domains (FIG. 29).

[0737] To produce a full and correctly assembled bispecific antibody that could specifically bind to at least two antigens, the variable heavy chain and variable light chain domains from a humanized anti human CD19 antibody (abbreviated VH(anti-hCD19) and VL(anti-hCD19) respectively, disclosed in the PCT Publication NO: WO 2010/095031), and the variable heavy chain and variable light chain domains from a humanized anti alpha 2 subunit of the human VLA-2 receptor (VLA2) antibody (abbreviated VH(anti-hVLA2) and VL(anti-hVLA2) respectively, disclosed in the PCT Publication NO: WO 2007/056858), were selected as inputs for expression and functional studies. The coding cDNAs for VH(anti-hVLA2) and VL(anti-hVLA2) domains were available from studies described in the PCT Publication NO: WO 2007/056858.

[0738] To create the four distinct chains of the bispecific antibody, two new immunoglobulin chains were created using PCR techniques: a first chain consisting of the variable heavy chain VH(anti-hVLA2) fused upstream of a human CH1(.gamma.1)-BT alpha His chain (example 1 and 2) (abbreviated VH(anti-hVLA2)-CH1-BT alpha His, SEQ ID NO: 66), and a second chain consisting of the variable light chain VL(hVLA2) fused upstream of the naturally occurring human Cc constant domain (abbreviated VL(anti-hVLA2)-CK, SEQ ID NO: 67). Following PCR assembly, both chain cDNAs were ligated independently into the modified pREP4 vector described example 1. The final two immunoglobulin chains needed to produce the complete bispecific antibody originated from example 5: a third chain consisting of the VH(anti-hCD19)-CH1 delta-BT beta c-myc chain (SEQ ID NO: 43), and a fourth chain consisting of the VL(anti-hCD19)-CK gamma chain (SEQ ID NO: 44). The resulting bispecific antibody having all the four distinct chains is abbreviated anti-hCD19_anti-hVLA2 bispecific antibody (SEQ ID NOs: 68, 69, 41, and 42).

[0739] All four vectors carrying the recombinant immunoglobulin chains were co-transfected into HEK293-EBNA cells and protein production was performed according to the procedure described in example 1. Small quantities of antibody species having a single specificity towards either the human VLA2 antigen (abbreviated anti-hVLA2 antibody) or the human CD19 antigen (abbreviated hCD19 antibody) were obtained by co-transfection of chain "one" with chain "two", and chain "three" with chain "four", respectively. These antibodies were later used as controls in FACS experiments (see below).

[0740] The production yields of transient transfections were about 18 mg/l post protein A purification (procedure according to example 1). To isolate fully assembled bispecific molecules from traces of homo-dimeric species, two affinity chromatographic steps were sequentially implemented. The first step was based on protein L affinity chromatography (Protein L resin from GenScript, Piscataway, N.J. USA; used according to the manufacturer's protocol), and made use of the specificity of protein-L towards the light chain variable domains originating from Kappa subgroup .kappa.1 and .kappa.3, but not .kappa.2 (Nilson B H et al., J Biol Chem, 267(4):2234-9 (1992)). Since the variable VL(anti-hVLA2) light chain domain belongs to the .kappa.2 subclass, and the variable VL(anti-hCD19) light chain domain belongs to the .kappa.1 subclass, this step allowed for the removal of any homo-dimeric antibody species lacking the variable VL(anti-hCD19) domain (see above, chain "four"). For the second step, the protein pool after protein L purification was buffer-exchanged into 50 mM Tris-HCl pH 9.0, 200 mM NaCl and loaded on a 1 ml HisTrap Ni.sup.2+-NTA affinity chromatography column operated on an .ANG.KTA Purifier (both from GE Healthcare Europe GmbH, Glattbrugg, Switzerland) at a flow rate of 0.6 ml/min. This step allowed for the removal of any homo-dimeric antibody species lacking the BT alpha chain (see above, chain "one").

[0741] To confirm the presence of the anti-hCD19_anti-hVLA2 bispecific antibody, a Western blot analysis was performed on sample aliquots isolated at different steps of the purification process. For detection, a horse radish peroxidase conjugated anti His tag (Sigma, Buchs, Switzerland) and anti c-myc tag (Roche Diagnostics (Schweiz) AG, Rotkreuz, Switzerland) antibodies were used. For staining, the peroxidase substrate SIGMA FAST 3,3'-Diaminobenzidine (Sigma, Buchs, Switzerland) was used. Comparison of two Western blots, one detected with the anti His tag antibody and one detected with the anti c-myc antibody confirmed the presence of the anti-hCD19_anti-hVLA2 bispecific antibody after the two purification steps (FIG. 30, lane 5 in panels B and C).

[0742] To confirm the dual specificity of the anti-hCD19 anti-hVLA2 bispecific antibody, two independent FACS binding experiments were designed. In both experiments, the bispecific antibody was tested for binding to one of its two cognate antigens displayed at the cell-surface of target cells while detecting with the affinity tag present on the chain having the second specificity. Since the anti-hCD19_anti-hVLA2 bispecific antibody specificities are towards both the human CD19 cell-surface protein and the alpha 2 subunit of the human VLA-2 receptor, the Raji cell line (a human Burkitt's lymphoma cell line, DSMZ clone NO: ACC319), and the HT1080 cell-line (human fibrosarcoma cell line, ATCC clone NO: CCL-121) were selected as target cells, respectively. Raji cells are strictly CD19.sup.+ and VLA2.sup.-, while HT1080 cells are VLA2.sup.+ and CD19.sup.- (data not shown), thereby preventing any false positives. Both cell lines were harvested from standard culture conditions and counted (viability >90% according to trypan blue exclusion). Cells were washed once in staining buffer: a PBS solution supplemented with 2.5% FBS from PAA (PAA Laboratories, Pasching, Austria; catalogue NO: A15-101) and 10% versene from GIBCO-Invitrogen AG (Basel, Switzerland, catalogue NO: 15040) prior to the staining procedure. Staining was performed on 2.times.10.sup.5 cells per condition, in V-bottom 96-well plates, on ice. Each incubation step was done in 50 .mu.l of staining buffer supplemented with 10 .mu.g/ml of anti-hCD19 anti-hVLA2 bispecific antibody and further incubated for 20 min. Cells were then washed twice with cold staining buffer and incubated 20 min with either a mouse anti His tag antibody (Sigma, Buchs, Switzerland; catalogue NO: A7058-1VL), or a mouse anti c-myc tag antibody (Roche Diagnostics (Schweiz) AG, Rotkreuz, Switzerland; catalogue NO: 11667149001), both at a concentration of 10 .mu.g/ml in staining buffer. A control sample with cells kept only in staining buffer was also made for both cell lines. For detection, cells were washed twice with staining buffer and incubated 20 min with a Phycoerythrin-conjugated mouse-anti-Human Ig Fc fragment specific antibody (eBioscience, San Diego, USA; distributor: eBioscience CBI Medicals PV, Baar, Switzerland; catalogue NO: 12-4998-82) used at a 1:200 dilution in staining buffer. Finally, cells were washed once with staining buffer and resuspended in 300 .mu.l of cold staining buffer and promptly analyzed by flow cytometry (CyAn.TM. ADP, Beckman Coulter International S.A., Nyon, Switzerland). Data were processed using the Flowjo.RTM. software (Tree Star, Inc., Ashland, Oreg., USA); histograms were plotted on live cells based on Forward and Side Scatter parameter gating. FIG. 31A shows the binding of the anti-hCD19_anti-hVLA2 bispecific antibody to Raji cells detected with a mouse-anti c-myc tag antibody, in this experiment the anti-hVLA2 antibody lacking the c-myc tag is used as a negative control. FIG. 31B shows the binding of the anti-hCD19_anti-hVLA2 bispecific antibody to Raji cells detected with a mouse-anti His tag antibody, in this experiment the anti-hVLA2 antibody is also used as a negative control, since it lacks the hCD19 specificity in spite of having the polyhistidine tag sequence. FIG. 31C shows the binding of the anti-hCD19_anti-hVLA2 bispecific antibody to HT1080 cells detected with a mouse-anti c-myc tag antibody, in this experiment the anti-hCD19 antibody is used as a negative control since it lacks the hVLA2 specificity in spite of having the c-myc tag sequence. FIG. 31D shows the binding of the anti-hCD9 anti-hVLA2 bispecific antibody to HT1080 cells detected with a mouse-anti His tag antibody, in this experiment the anti-hCD19 antibody lacking the polyhistidine tag sequence is used as a negative control. When taken together these four independent binding experiments show that when engaging target cells with one FAB arm, the anti-hCD19_anti-hVLA2 bispecific antibody is always detectable via the tag located on the opposite Fc subunit (carrying the other FAB arm, see FIGS. 31B and 31C). This demonstrates the identity of the anti-hCD19_anti-hVLA2 as being a full bispecific antibody consisting of four different chains correctly assembled into two distinct FAB fragments each having a unique specificity brought together by a hetero-dimeric Fc region.

[0743] The thermal stability of the anti-hCD19_anti-hVLA2 bispecific antibody was assessed using calorimetric measurements (DSC) (see example 2 for methods; the protein was used at a concentration of 0.57 mg/ml in PBS). FIG. 32 shows a thermogram of the anti-hCD19_anti-hVLA2 bispecific antibody exhibiting three sharp transitions at 70.9, 76.6 and 82.8.degree. C., none of which are below 70.degree. C. thereby implying a stable immunoglobulin molecule (example 2). It is important to note that no sharp decrease after the heat absorption peak was recorded, indicating that no precipitation or aggregate formation occurred after thermal unfolding (Liu H et al., Immunol Lett, 106(2):144-53 (2006)). Since the BT alpha His_BT beta hetero-dimeric Fc fragment from example 2 displayed a single sharp melting transition at 70.degree. C., it is possible to conclude that the melting transitions for both FAR arms have a mid-point superior to 75.degree. C. as expected for a properly assembled and stable FAB fragments (Garber E and Demarest S J, Biochem Biophys Res Commun, 355(3):751-7 (2007)). In conclusion, this thermal unfolding study shows that the anti-hCD19_anti-hVLA2 bispecific antibody is stable.

[0744] This example also shows that when combined, two or more novel hetero-dimeric pairs of constant domains having engineered protein-protein interfaces derived form the human T-cell receptor constant domains can be used as building blocks to create novel bispecific antibodies which are stable and suitable for therapeutic use in humans.

Example 10: Construction of Immunoglobulin Fc Hetero-Dimer Variants with Mixed Gamma Immunoglobulin Isotype Backgrounds

[0745] This example demonstrates that two human CH3 domains, one domain derived from IGHG1 and the second domain derived from IGHG3 having mutations in the protein-protein interface of their CH3 domains carefully selected from the T-cell receptor (TCR) constant domain alpha and beta (as described in example1), assemble into a hetero-dimeric Fc molecule.

[0746] Mutations were derived from the analysis of an overlay of the crystal structure of the human LC13 TCR molecule (PDB code IKGC; Bernstein F C et al., Eur J Biochem, 80(2):319-24 (1977)) with the crystal structure of the Fc fragment from human IGHG1 (PDB code 1H3Y) as described in example 1. Since human IGHG1 and IGHG3 CH3 domains only differ at position 384, 392, 397, 422, 435, and 436 (EU numbering), their protein-protein interfaces are identical excepting for the residue at position 392, and allow for the design of a mixed isotype hetero-dimeric immunoglobulin based on the 3D equivalent positions described in example 1.

[0747] The hetero-dimeric immunoglobulin described in this example consists of the assembly of two Fc chain variants: one chain from human IGHG3 origin having mutations in the protein-protein interface of its CH3 domain carefully selected from the TCR constant domain alpha protein-protein interface (referred as B1 alpha IGHG3 chain) (SEQ ID NO: 73), and one chain from human IGHG1 origin having mutations in the protein-protein interface of its CH3 domain carefully selected from the TCR constant domain beta protein-protein interface (referred as BT beta F405A chain) (SEQ ID NO: 74). To generate a difference in SDS-PAGE mobility, the latter Fc chain variant was fused to a variable light-chain kappa domain antibody (abbreviated VL) resulting in an engineered immunoglobulin chain referred as VL-BT beta F405A chain (SEQ ID NO: 75). The engineered C1L3 domain from IGHG3 origin described herein is abbreviated CH3-BT alpha IGHG3 domain, and has SEQ ID NO: 76. The VL-BT beta F405A chain encompasses the CH3-Br beta F405A domain described in example 2 (SEQ ID NO: 14). The hetero-dimeric immunoglobulin consisting of the assembly of the two aforementioned chains is abbreviated BT alpha IGHG3_VL-BT beta F405A hetero-dimer.

[0748] cDNA encoding the VL-BT beta F405A chain was built as described in example 2, cDNA encoding the BT alpha IGHG3 chain encompassed the CH3-BT alpha IGHG3 domain coding sequence (SEQ ID NO: 77) which was originally synthesized by GENEART AG (Regensburg, Germany). Human IGHG1 hinge and CH2 domain coding sequences were subsequently added upstream to this synthesized fragment using standard PCR assembly methods. Each chain encoding PCR product was digested, purified, and ligated independently into the modified pREP4 vector mentioned previously (example 1). The two resulting sequence-verified recombinant vectors were then co-transfected into HEK293-EBNA cells as described in example 1. Protein production and purification were also according to example 1. For the BT alpha IGHG3_VL-BT beta F405A hetero-dimer, a production yield of 10 mg/l was obtained.

[0749] To assess the proportion of hetero-dimer to homo-dimer in the protein-A purified material, the relative ratios of the different species were quantified by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands according to the procedure described in example 1. Post protein-A purification the BT alpha IGHG3_VL-BT beta F405A hetero-dimer is the main species produced, followed by the VL-BT beta F405A_VL-BT beta F405A homo-dimer, and the VL-BT beta F405A chain (half molecule) (FIG. 33; lane 1). Under reducing conditions, the BT alpha IGHG3_VL-BT beta F405A hetero-dimer breaks down into the two expected molecular weight bands for the BT alpha IGHG3 and the VL-BT beta F405A chains (half molecules) (FIG. 33; lane 2). FIG. 34 shows that BT alpha IGHG3_VL-BT beta F405A hetero-dimer represents at least 91% of the protein-A purified material.

Example 11: Construction of Immunoglobulin Fc Hetero-Dimer Variants with Mixed Immunoglobulin Class Backgrounds

[0750] This example demonstrates that two human Fc chains, derived from IGHG1 having mutations in the protein-protein interface of their CH3 domains carefully selected from the protein-protein interface of the CH3 domains from IGHA1 or IGHA2, assemble into a hetero-dimeric Fc molecule, thereby demonstrating that protein-protein interfaces from two different pairs of homo-dimeric immunoglobulin domains can be combined to create a novel hetero-dimeric protein-protein interface pair which upon grafting onto a naturally occurring pair of homo-dimeric immunoglobulin domains induce domain hetero-dimerization. Note that the scope of the method described herein is not limited to IGHA1, but has broad application using all type of homo-dimeric immunoglobulin domains. IGHA1 and IGHA2 have identical CH3 domain amino acid sequences, and IGHA1 CH3 amino acid sequence (SEQ ID NO: 96) used herein is fully interchangeable with IGHA2 CH3 amino acid sequence (SEQ ID NO: 97).

[0751] Mutations were derived from the analysis of the CH3 domain protein-protein interface interactions of the crystal structure of human IGH1 and IGHA1 Fc fragments (Krapp S et al., J Mol Biol, 325(5):979-89 (2003) and Herr A B et al., Nature, 423(6940):614-20 (2003), respectively). The IGHG1 and IGHA1 Fc 3D structures were retrieved from the Protein Data Bank (PDB code 1H3Y and IOW0 respectively; www.pdb.org; Bernstein F C et al., Eur J Biochem, 80(2):319-24 (1977)) and further analyzed as described in example 1.

[0752] The hetero-dimeric immunoglobulin described in this example consists of the assembly of two Fc chain variants: one human IGHG1 Fc chain thereof having mutations in the protein-protein interface of its CH3 domain derived from a selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human IGHA1 CH3 constant domain pair (designated "Fc IGHAG" chain), and a second engineered human IGHG1 Fc chain thereof having mutations in the protein-protein interface of its CH3 domain derived from a selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human IGHA1 CH3 constant domain pair (designated "Fc IGHGA" chain), wherein the selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human IGHA1 CH3 constant domain pair is different in each engineered CH3 domain. More specifically, the "Fc IGHAG" chain consists of an immunoglobulin Fc chain from human IGHG1 having a CH3 domain with the following substitutions (abbreviated CH3 IGHAG domain; SEQ ID NO: 78): Q347E, Y349H, K370R, V397R, D399E, F405A, and Y407T (EU numbering); and conversely, the "Fc IGHGA" consists of an immunoglobulin Fc chain from human IGHG1 having a CH3 domain with the following substitutions (abbreviated CH3 IGHGA domain; SEQ ID NO: 79): S364T, K392L, T394W, K409I, and T411R (EU numbering). To generate a difference in SDS-PAGE mobility, one Fc chain variant was fused to a variable light-chain kappa domain antibody (abbreviated VL). The different PCR steps gave two final chains: the Fc IGHAG chain (SEQ ID NO: 80), and the VL-Fc IGHGA chain (SEQ ID NO: 81). The hetero-dimeric immunoglobulin construct described herein resulting from the assembly of these two chains is designated Fc IGHAG_VL-Fc IGHGA hetero-dimer.

[0753] cDNA coding sequences for the CH3 IGHGA, and CH3 IGHAG domains (SEQ ID NO: 82 and 83, respectively) were synthesized by GENEART AG (Regensburg, Germany), and used to create their respective engineered chains by PCR assembly methods. Each chain encoding PCR product was digested, purified, and ligated independently into the modified pREP4 vector mentioned previously (example 1). The two resulting sequence-verified recombinant vectors were then co-transfected into HEK293-EBNA cells as described in example 1. Protein production and purification were also according to example 1. For the Fc IGHAG_VL-Fc IGHGA hetero-dimer, a production yield of 12 mg/l was obtained.

[0754] To assess the proportion of hetero-dimer to homo-dimer in the protein-A purified material, the relative ratios of the different species were quantified by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands according to the procedure described in example 1. Post protein-A purification, the Fc IGHAG_VL-Fc IGHGA hetero-dimer is the main species produced followed by the VL-Fc IGHGA_VL-Fc IGHGA homo-dimer and single chains (Fc IGHAG chain and VL-Fc IGHGA chains, i.e. half molecules) (FIG. 35, lane 1). Under reducing conditions, the Fc IGHAG_VL-Fc IGHGA hetero-dimer breaks down into the two expected molecular weight bands for the Fc IGHAG and the VL-Fc IGHGA chains (FIG. 35, lane 2). FIG. 36 shows that the Fc IGHAG_VL-Fc IGHGA hetero-dimer represents at least 54% of the protein-A purified material.

Example 12: Construction of Immunoglobulin Fc Hetero-Dimer Variants with Different Immunoglobulin Class Backgrounds

[0755] 12.1 Construction of a Hetero-Dimer Variant with a Chimeric IGHG1-IGHM Immunoglobulin Class Background

[0756] This example demonstrates that two chains, each consisting of a human IGHG1 hinge, a human IGHG1 CH2 domain and an engineered human IGHM CH4 domain, wherein one chain has mutations in the protein-protein interface of its IGHM CH4 domain carefully selected from 3D equivalent positions of the protein-protein interface of the T-cell receptor (TCR) constant domain alpha and the second chain has mutations in the protein-protein interface of its IGHM CH4 domain carefully selected from 3D equivalent positions of the protein-protein interface of the TCR constant domain beta, assemble into a hetero-dimeric immunoglobulin; the 3D equivalent positions are identical to the ones described in example 1.

[0757] Specifically, the hetero-dimeric immunoglobulin described in this example consists of the assembly of two Fc chain variants: one engineered chain comprising an engineered IGHM CH4 domain wherein its protein-protein interface is substituted with a selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human TCR constant domain alpha (designated "BT alpha IGHM-4" chain), and a second engineered chain comprising an engineered IGHM CH4 domain wherein its protein-protein interface is substituted with a selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human TCR constant domain beta (designated "BT beta IGHM-4" chain). More specifically, the BT alpha IGHM-4 chain encompasses a CH4 domain from human IGHM with the following substitutions (abbreviated IGHM CH4 alpha domain; SEQ ID NO: 84): T20K, T22V, V79Y, F85.1S, H86V, I88W, and T90N IMGT.RTM. numbering); and conversely, the BT beta IGHM-4 chain encompasses of a CH4 domain from human IGHM with the following substitutions (abbreviated IGHM CH4 beta domain; SEQ ID NO: 85): D3E, Y5A, L7F, T22V, S81D, M84L, F85.1A, H86S, I88R, and T90R (IMGT numbering).

[0758] cDNA coding sequences for the IGHM CH4 alpha domain and IGHM CH4 beta domain (SEQ ID NO: 86 and 87, respectively) were synthesized by GENEART AG (Regensburg, Germany), and used to create their respective engineered chains by PCR assembly methods. A short stretch of six histidine residues was appended at the C-terminus of the BT alpha IGHM-4 chain (abbreviated His) by including a polyhistidine coding sequence in the PCR anti-sense oligonucleotide. To generate a difference in SDS-PAGE mobility, one Fc chain variant was fused to a variable light-chain kappa domain antibody (abbreviated VL). The different PCR steps gave two final chains: the BT alpha IGHM-4 His chain (SEQ ID NO: 88), and the VL-BT beta IGHM-4 chain (SEQ ID NO: 89) (FIG. 37 labelled A and FIG. 37 labelled B). The hetero-dimeric immunoglobulin construct described herein resulting from the assembly of these two chains is designated BT alpha IGHM-4 His_VL-BT IGHM-4 beta hetero-dimer.

[0759] Each chain encoding PCR product was digested, purified, and ligated independently into the modified pREP4 vector mentioned previously. The two resulting sequence-verified recombinant vectors were subsequently co-transfected into HEK293-EBNA cells as described in example 1. Protein expression was also according to example 1. Purification was performed using protein-L affinity agarose (GenScript USA Inc., Piscataway, N.J., USA). The proportion of hetero-dimer to homo-dimer in the cell-culture supernatants and protein-L purified preparations were assessed by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands (procedure according to example 1).

[0760] Post protein-L purification, the BT alpha IGHM-4 His_VL-BT IGHM-4 beta hetero-dimer is isolated without any contaminants (FIG. 38, lane 3, labelled as A), representing 100% of the protein-L eluted material as measured by scanning densitometry analysis of the non-reduced SDS-PAGE band (FIG. 39). Under reducing conditions, the BT alpha (IGM-4 His_VL-BT beta IGHM-4 hetero-dimer band breaks down into the two expected molecular weight bands for the BT alpha IGHM-4 His and VL-BT beta IGHM-4 chains (half molecules) (FIG. 38, lane 4).

12.2 Construction of a Hetero-Dimer with a Chimeric IGHG1-IGHE Immunoglobulin Class Background

[0761] This example demonstrates that two chains, each consisting of a human IGHG1 hinge, a human IGHG1 CH2 domain and an engineered human IGHE CH4 domain, wherein one chain has mutations in the protein-protein interface of its IGHE CH4 domain carefully selected from 3D equivalent positions of the protein-protein interface of the T-cell receptor (TCR) constant domain alpha and the second chain has mutations in the protein-protein interface of its IGHE CH4 domain carefully selected from 3D equivalent positions of the protein-protein interface of the TCR constant domain beta, assemble into a hetero-dimeric immunoglobulin; the 3D equivalent positions are identical to the ones described in example 1.

[0762] Specifically, the hetero-dimeric immunoglobulin described in this example consists of the assembly of two Fc chain variants: one engineered chain comprising an engineered IGHE CH4 domain wherein its protein-protein interface is substituted with a selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human TCR constant domain alpha (designated "BT alpha IGHE-4" chain), and a second engineered chain comprising an engineered IGHE CH4 domain wherein its protein-protein interface is substituted with a selected group of 3D equivalent positions found in the protein-protein interface of the naturally occurring human TCR constant domain beta (designated "BT beta IGHE-4" chain). More specifically, the BT alpha IGHE-4 chain encompasses a CH4 domain from human IGHE with the following substitutions (abbreviated IGHE CH4 alpha domain; SEQ ID NO: 90): T20K, A22V, Q26T, S79Y, F85.1S, F86V, R88W, and E90N (IMGT numbering); and conversely, the BT beta IGHGE4 chain encompasses a CH4 domain from human IGHE with the following substitutions (abbreviated IGHE CH4 beta domain; SEQ ID NO: 91): Y5A, A22V, Q26T, T81D, R84L, T84.2E, F85.1A, F86S, and E90R (IMGT numbering).

[0763] Similarly to example 12.1, cDNA coding sequences for the IGHE CH4 alpha and IGHE CH4 beta domains (SEQ ID NO: 92 and 93, respectively) were synthesized by GENEART AG (Regensburg, Germany), and used to create their respective engineered chains by PCR assembly methods. A short stretch of six histidine residues was appended at the C-terminus of the BT alpha IGHE-4 chain (abbreviated His) by including a polyhistidine coding sequence in the PCR anti-sense oligonucleotide. To generate a difference in SDS-PAGE mobility, one FE chain variant was fused to a variable light-chain kappa domain antibody (abbreviated VL). The different PCR steps gave two final chains; the BT alpha IGHE-4 His chain (SEQ ID NO: 94), and the VL-BT IGHE-4 beta chain (SEQ ID NO: 95) (FIG. 37 labelled C and FIG. 37 labelled D). The hetero-dimeric immunoglobulin construct described herein resulting from the assembly of these two chains is designated BT alpha IGHE-4 His_VL-BT IGHE-4 beta hetero-dimer.

[0764] Each chain encoding PCR product was digested, purified, and ligated independently into the modified pREP4 vector mentioned previously. The two resulting sequence-verified recombinant vectors were subsequently co-transfected into HEK293-EBNA cells as described in example 1. Protein production and purification were also according to example 1. The proportion of hetero-dimer to homo-dimer in the protein-A purified preparations were assessed by scanning densitometry analysis of the non-reduced SDS-polyacrylamide (4-12%) gel bands (procedure according to example 1).

[0765] Post protein-A purification, the hetero-dimer is isolated (FIG. 40, lane 1, labelled as A) with a hetero-dimerization rate of 10% (FIG. 41, peak 2), and bands corresponding to the BT alpha IGHE-4 His homo-dimer (FIG. 40, lane 1, labelled as B), the VL-BT beta IGHE-4 chain (half molecule) (FIG. 40, lane 1, labelled as C), and the BT alpha IGHE-4 His chain (half molecule) (FIG. 40, lane 1, labelled as D) are observed. Under reducing conditions, the BT alpha IGHE-4 His_VL-BT beta IGHE-4 hetero-dimer breaks down into the two expected molecular weight bands for the BT alpha IGHE-4 His and VL-BT beta IGHE-4 chains (half molecules) (FIG. 40, lane 2).

Sequence CWU 1

1

124191PRTHomo sapiensMISC_FEATURE(1)..(91)T cell receptor constant domain alpha 1Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys1 5 10 15Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr 20 25 30Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 35 40 45Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala 50 55 60Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser65 70 75 80Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 85 902128PRTHomo sapiensMISC_FEATURE(1)..(128)T cell receptor constant domain beta (Homo sapiens) 2Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu65 70 75 80Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90 95Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 100 105 110Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 115 120 1253227PRTArtificial SequenceSynthetic polypeptide 3Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Lys 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys2254227PRTArtificial SequenceSynthetic polypeptide 4Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125Ala Thr Phe Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro 165 170 175Leu Leu Glu Ser Asp Gly Ser Phe Cys Leu Ser Ser Arg Leu Arg Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys2255108PRTArtificial SequenceSynthetic polypeptide 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 1056335PRTArtificial SequenceSynthetic polypeptide 6Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu 245 250 255Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser 275 280 285Asp Gly Ser Phe Cys Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 3357233PRTArtificial SequenceSynthetic polypeptide 7Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Lys 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys His His His His His His225 2308321DNAArtificial SequenceSynthetic polynucleotide 8ggccagccca gagaacccca ggtgtacacc ctgcccccca gcagagatga gctgaccaag 60aaccaggtca agctcgtgtg cctggtcacc ggcttctacc ccagcgatat cgccgtggag 120tgggagagca acggccagcc tgaaaacaac tactacacca ccccccctgt gctggacagc 180gacggcagct tcagcctggt gtcctggctg aacgtggaca agagccggtg gcagcagggc 240aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 300ctgagcctgt ctcctggcaa g 3219321DNAArtificial SequenceSynthetic polynucleotide 9ggacagccca gggaacctga ggtggccaca ttcccaccta gccgggacga gctgacaaaa 60aatcaggtca ccctcgtctg tctcgtgacc ggcttttacc cttccgacat tgccgtggaa 120tgggaatcca atgggcagcc cgagaacaat tacaagacag acccccccct gctggaatcc 180gatggcagct tctgcctgag cagccggctg cgggtggaca agtccagatg gcagcagggg 240aatgtctttt cctgctccgt catgcatgaa gccctccaca atcattatac acagaaaagc 300ctgagcctga gccccggcaa g 32110699DNAArtificial SequenceSynthetic polynucleotide 10gacaaaactc acacatgccc cccctgccct gcccctgagc tgctgggcgg accctccgtg 60ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccggacccc cgaggtgacc 120tgcgtggtgg tggacgtgag ccacgaggac cctgaggtga agttcaattg gtacgtggac 180ggcgtggagg tgcacaacgc caagaccaag ccccgggagg aacagtacaa cagcacctac 240cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggaatacaag 300tgcaaggtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatctc caaagccaaa 360ggccagccca gagaacccca ggtgtacacc ctgcccccca gcagagatga gctgaccaag 420aaccaggtca agctcgtgtg cctggtcacc ggcttctacc ccagcgatat cgccgtggag 480tgggagagca acggccagcc tgaaaacaac tactacacca ccccccctgt gctggacagc 540gacggcagct tcagcctggt gtcctggctg aacgtggaca agagccggtg gcagcagggc 600aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660ctgagcctgt ctcctggcaa gcatcaccat caccatcac 699111005DNAArtificial SequenceSynthetic polynucleotide 11gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgggtgacc 60atcacctgcc gggccagcca gagcatcagc tcctacctga actggtatca gcagaagccc 120ggcaaggccc ccaagctgct gatctatgct gcctcctctc tccagagcgg cgtgcccagc 180cggttttccg ggtctgggtc cgggacagat ttcaccctga ccatcagcag cctccagccc 240gaggatttcg ccacctacta ctgccagcag agctacagca cccccaacac cttcggccag 300ggaacaaagg tggagatcaa gcgggacaaa actcacactt gcccaccgtg cccagcacct 360gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 420atctcccgga cccctgaggt cacctgcgtg gtggtggacg tgagccacga ggaccctgag 480gtgaagttca attggtacgt ggacggcgtg gaggtgcaca acgccaagac caagccccgg 540gaggaacagt acaacagcac ctaccgggtg gtgtccgtgc tgaccgtgct gcaccaggac 600tggctgaacg gcaaggaata caagtgcaag gtctccaaca aggccctgcc tgcccccatc 660gaaaagacca tcagcaaggc caagggacag cccagggaac ctgaggtggc cacattccca 720cctagccggg acgagctgac aaaaaatcag gtcaccctcg tctgtctcgt gaccggcttt 780tacccttccg acattgccgt ggaatgggaa tccaatgggc agcccgagaa caattacaag 840acagaccccc ccctgctgga atccgatggc agcttctgcc tgagcagccg gctgcgggtg 900gacaagtcca gatggcagca ggggaatgtc ttttcctgct ccgtcatgca tgaagccctc 960cacaatcatt atacacagaa aagcctgagc ctgagccccg gcaag 100512227PRTArtificial SequenceSynthetic polypeptide 12Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22513107PRTArtificial SequenceSynthetic polypeptide 13Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Lys Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Tyr Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Ser Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10514107PRTArtificial SequenceSynthetic polypeptide 14Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly Ser Phe 50 55 60Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10515107PRTArtificial SequenceSynthetic polypeptide 15Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly Ser Phe 50 55 60Cys Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10516107PRTArtificial SequenceSynthetic polypeptide 16Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Asp Pro Pro

Leu Leu Glu Ser Asp Gly Ser Phe 50 55 60Ser Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10517236PRTArtificial SequenceSynthetic polypeptide 17Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125Ala Thr Phe Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro 165 170 175Leu Leu Glu Ser Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala225 230 23518227PRTArtificial SequenceSynthetic polypeptide 18Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Trp Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22519227PRTArtificial SequenceSynthetic polypeptide 19Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Trp Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22520227PRTArtificial SequenceSynthetic polypeptide 20Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Val Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Trp Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22521227PRTArtificial SequenceSynthetic polypeptide 21Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Trp Leu Asn Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22522227PRTArtificial SequenceSynthetic polypeptide 22Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22523227PRTArtificial SequenceSynthetic polypeptide 23Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Lys 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Trp Leu Asn Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22524335PRTArtificial SequenceSynthetic polypeptide 24Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Ala Leu Ser Ser Lys Leu Thr Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33525335PRTArtificial SequenceSynthetic polypeptide 25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Ala Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33526335PRTArtificial SequenceSynthetic polypeptide 26Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Phe Leu Ser Ser Arg Leu Thr Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33527335PRTArtificial SequenceSynthetic polypeptide 27Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Arg Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33528335PRTArtificial SequenceSynthetic polypeptide 28Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Thr Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33529335PRTArtificial SequenceSynthetic polypeptide 29Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Ser Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33530227PRTArtificial SequenceSynthetic polypeptide 30Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22531335PRTArtificial SequenceSynthetic polypeptide 31Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 3353287PRTArtificial SequenceSynthetic polypeptide 32Arg Ser Gln Pro His Thr Lys Pro Ser Val Phe Val Met Lys Asn Gly1 5 10 15Thr Asn Val Ala Cys Leu Val Lys Glu Phe Tyr Pro Lys Asp Ile Arg 20 25 30Ile Asn Leu Val Ser Ser Lys Lys Ile Thr Glu Phe Asp Pro Ala Ile 35 40 45Val Ile Ser Pro Ser Gly Lys Tyr Asn Ala Val Lys Leu Gly Lys Tyr 50 55 60Glu Asp Ser Asn Ser Val Thr Cys Ser Val Gln His Asp Asn Lys Thr65 70 75 80Val His Ser Thr Asp Phe Glu 8533101PRTArtificial SequenceSynthetic polypeptide 33Val Ser Pro Lys Pro Thr Ile Phe Leu Pro Ser Ile Ala Glu Thr Lys1 5 10 15Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu Leu Glu Lys Phe Phe Pro 20 25 30Asp Val Ile Lys Ile His Trp Glu Glu Lys Lys Ser Asn Thr Ile Leu 35 40 45Gly Ser Gln Glu Gly Asn Thr Met Lys Thr Asn Asp Thr Tyr Met Lys 50 55 60Phe Ser Trp Leu Thr Val Pro Glu Lys Ser Leu Asp Lys Glu His Arg65 70 75 80Cys Ile Val Arg His Glu Asn Asn Lys Asn Gly Val Asp Gln Glu Ile 85 90 95Ile Phe Pro Pro Ile 10034227PRTArtificial SequenceSynthetic polypeptide 34Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Met Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Asn 130 135 140Leu Ala Cys Leu Val Lys Glu Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Phe Thr Ala Pro Pro 165 170 175Val Leu Ser Ser Asp Gly Ser Phe Asn Leu Val Ser Leu Leu Lys Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22535227PRTArtificial SequenceSynthetic polypeptide 35Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Lys Val 115 120 125Thr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr 130 135 140Leu Leu Cys Leu Val Glu Lys Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Gly Pro Pro 165 170 175Met Leu Asp Ser Asp Gly Ser Phe Met Leu Phe Ser Trp Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln

Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22536335PRTArtificial SequenceSynthetic polypeptide 36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Lys Val Thr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Leu Cys Leu 245 250 255Val Glu Lys Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Gly Pro Pro Met Leu Asp Ser 275 280 285Asp Gly Ser Phe Met Leu Phe Ser Trp Leu Thr Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33537321DNAArtificial SequenceSynthetic polynucleotide 37ggccagccca gagaacccca ggtgtacacc atgcccccca gcagggacga gctgaccaag 60aaccaggtca acctggcctg cctggtcaaa gagttctacc ccagcgatat cgccgtggaa 120tgggagagca acggccagcc tgagaacaac tacttcaccg cccctcccgt gctgagcagc 180gacggcagct tcaacctggt gtccctgctg aaggtggaca agagccggtg gcagcagggc 240aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 300ctgagcctga gccccggaaa g 32138321DNAArtificial SequenceSynthetic polynucleotide 38ggccagcccc gcgagcccaa agtgacaacc ctgcccccca gccgggacga gctgaccaag 60aatcaggtca cactgctgtg cctggtggaa aagttctacc ccagcgatat cgccgtggaa 120tgggagagca acggccagcc cgagaacaac tacaagaccg gccctcccat gctggacagc 180gacggcagct tcatgctgtt cagctggctg accgtggaca agagccggtg gcagcagggc 240aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 300ctgagcctga gccccggaaa g 3213998PRTArtificial SequenceSynthetic polypeptide 39Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Met Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Asn Leu Ala Cys Leu Val Lys Glu Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val Phe Thr Ala Pro Ala Val Leu Ser Ser Ser Gly Leu Tyr Asn 50 55 60Leu Val Ser Leu Val Lys Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val40107PRTArtificial SequenceSynthetic polypeptide 40Arg Thr Val Ala Ala Pro Lys Val Thr Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Thr Val Leu Cys Leu Leu Glu Lys Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Gly Val Met Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Met Leu Phe Ser Trp Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10541461PRTArtificial SequenceSynthetic polypeptide 41Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Met Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Asn 130 135 140Leu Ala Cys Leu Val Lys Glu Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val Phe Thr Ala Pro Ala Val 165 170 175Leu Ser Ser Ser Gly Leu Tyr Asn Leu Val Ser Leu Val Lys Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala 340 345 350Thr Phe Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu 355 360 365Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu385 390 395 400Leu Glu Ser Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 450 455 46042214PRTArtificial SequenceSynthetic polypeptide 42Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Ile Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Leu Pro Tyr 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Lys Val Thr Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Thr Val Leu Cys Leu Leu Glu Lys Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Gly Val Met Glu Gln Asp Ser Lys Asp Ser Thr Tyr Met Leu Phe 165 170 175Ser Trp Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210431383DNAArtificial SequenceSynthetic polynucleotide 43caggtccagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggcgt gagcctgccc gactacggcg tgagctgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtg atctggggct ccgagacaac ctactacaac 180agcgccctga agagccgatt caccatctcc agagacaatt ccaagaacac gctgtatttg 240caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgaa gcactactac 300tacggcggca gctacgccat ggactactgg ggccagggaa ccctggtcac cgtctcctca 360gcgtcgacca agggccccag cgtgttcccg atggccccca gcagcaagag caccagcggc 420ggcacagcca acctggcctg cctggtgaag gagtacttcc ccgagcccgt gaccgtgtcc 480tggaactctg gagccctgac ctccggcgtg ttcaccgccc ccgccgtgct ctccagcagc 540ggcctgtaca acctggtgag cctcgtgaag gtgcccagca gcagcctggg aacccagacc 600tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc 660aagagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag gacagcccag ggaacctgag gtggccacat tcccacctag ccgggacgag 1080ctgacaaaaa atcaggtcac cctcgtctgt ctcgtgaccg gcttttaccc ttccgacatt 1140gccgtggaat gggaatccaa tgggcagccc gagaacaatt acaagacaga cccccccctg 1200ctggaatccg atggcagctt cgccctgagc agccggctgc gggtggacaa gtccagatgg 1260cagcagggga atgtcttttc ctgctccgtc atgcatgaag ccctccacaa tcattataca 1320cagaaaagcc tgagcctgtc tcctggcaag gaacaaaaac tcatctcaga agaggatctg 1380aat 138344642DNAArtificial SequenceSynthetic polynucleotide 44gatattcaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaaacca 120gggaaagcca tcaagctcct gatctatcac accagccggc tgcacagcgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcagcaa ggcgccacac tgccctacac cttcggccct 300gggaccaaag tggatatcaa acgtacggtg gccgctccca aggtgaccat cttccccccc 360agcgacgagc agctgaagag cggcaccgcc accgtgctgt gcctgctgga gaagttctac 420ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tccagagcgg caacagccag 480gaaggcgtca tggagcagga cagcaaggac tccacctaca tgctgttcag ctggctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gc 64245294DNAArtificial SequenceSynthetic polynucleotide 45gcgtcgacca agggccccag cgtgttcccg atggccccca gcagcaagag caccagcggc 60ggcacagcca acctggcctg cctggtgaag gagtacttcc ccgagcccgt gaccgtgtcc 120tggaactctg gagccctgac ctccggcgtg ttcaccgccc ccgccgtgct ctccagcagc 180ggcctgtaca acctggtgag cctcgtgaag gtgcccagca gcagcctggg aacccagacc 240tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtg 29446321DNAArtificial SequenceSynthetic polynucleotide 46cgtacggtgg ccgctcccaa ggtgaccatc ttccccccca gcgacgagca gctgaagagc 60ggcaccgcca ccgtgctgtg cctgctggag aagttctacc cccgggaggc caaggtgcag 120tggaaggtgg acaacgccct ccagagcggc aacagccagg aaggcgtcat ggagcaggac 180agcaaggact ccacctacat gctgttcagc tggctgaccc tgagcaaggc cgactacgag 240aagcacaagg tgtacgcctg cgaggtgacc caccagggcc tgtccagccc cgtgaccaag 300agcttcaacc ggggcgagtg c 32147107PRTArtificial SequenceSynthetic polypeptide 47Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 1054898PRTArtificial SequenceSynthetic polypeptide 48Ala Ser Thr Lys Gly Pro Gln Val Tyr Pro Leu Ala Pro Ser Ser Asp1 5 10 15Glu Thr Ser Gly Gly Gln Ala Ser Leu Thr Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val Lys Thr Thr Pro Ala Val Leu Asp Ser Ser Gly Leu Tyr Phe 50 55 60Leu Tyr Ser Lys Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val49107PRTArtificial SequenceSynthetic polypeptide 49Arg Thr Val Ala Ala Pro Gln Val Tyr Ile Leu Pro Pro Ser Asp Asp1 5 10 15Glu Leu Lys Ser Gly Thr Ala Ser Val Thr Cys Leu Leu Lys Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Lys Glu Thr Val Val Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Phe Leu Tyr Ser Lys Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10550461PRTArtificial SequenceSynthetic polypeptide 50Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Gln Val 115 120 125Tyr Pro Leu Ala Pro Ser Ser Asp Glu Thr Ser Gly Gly Gln Ala Ser 130 135 140Leu Thr Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val Lys Thr Thr Pro Ala Val 165 170 175Leu Asp Ser Ser Gly Leu Tyr Phe Leu Tyr Ser Lys Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala 340 345 350Thr Phe Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu 355 360 365Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu385 390 395 400Leu Glu Ser Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 450 455 46051214PRTArtificial SequenceSynthetic polypeptide 51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Ile Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Leu Pro Tyr 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Gln Val Tyr Ile Leu Pro Pro Ser Asp Asp Glu Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Thr Cys Leu Leu Lys Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Lys145 150 155 160Glu Thr Val Val Glu Gln Asp Ser Lys Asp Ser Thr Tyr Phe Leu Tyr 165 170 175Ser Lys Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210521383DNAArtificial SequenceSynthetic polynucleotide 52caggtccagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggcgt gagcctgccc gactacggcg tgagctgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtg atctggggct ccgagacaac ctactacaac 180agcgccctga agagccgatt caccatctcc agagacaatt ccaagaacac gctgtatttg 240caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgaa gcactactac 300tacggcggca gctacgccat ggactactgg ggccagggaa ccctggtcac cgtctcctca 360gccagcacca agggccccca ggtgtaccct ctggccccca gcagcgacga gacaagcgga 420ggccaggcca gcctgacctg cctggtgaaa gactacttcc ccgagcccgt gaccgtgtcc 480tggaactctg gcgccctgac cagcggcgtg aaaaccaccc ctgctgtgct ggacagcagc 540ggcctgtact tcctgtacag caaagtgacc gtgcctagca gcagcctggg cacccagacc 600tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa agtggaaccg 660aaaagctgcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag gacagcccag ggaacctgag gtggccacat tcccacctag ccgggacgag 1080ctgacaaaaa atcaggtcac cctcgtctgt ctcgtgaccg gcttttaccc ttccgacatt 1140gccgtggaat gggaatccaa tgggcagccc gagaacaatt acaagacaga cccccccctg 1200ctggaatccg atggcagctt cgccctgagc agccggctgc gggtggacaa gtccagatgg 1260cagcagggga atgtcttttc ctgctccgtc atgcatgaag ccctccacaa tcattataca 1320cagaaaagcc tgagcctgtc tcctggcaag gaacaaaaac tcatctcaga agaggatctg 1380aat 138353642DNAArtificial SequenceSynthetic polynucleotide 53gatattcaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaaacca 120gggaaagcca tcaagctcct gatctatcac accagccggc tgcacagcgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcagcaa ggcgccacac tgccctacac cttcggccct 300gggaccaaag tggatatcaa aagaaccgtg gctgctcctc aggtgtacat cctgcctccc 360agcgacgatg agctgaagtc tggcaccgcc agcgtgacat gtctgctgaa gaacttctac 420ccccgcgagg ccaaggtgca gtggaaagtg gacaacgccc tgcagagcgg caacagcaaa 480gaaaccgtgg tggaacagga cagcaaggac tccacctact ttctgtactc caagctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gc 64254294DNAArtificial SequenceSynthetic polynucleotide 54gccagcacca agggccccca ggtgtaccct ctggccccca gcagcgacga gacaagcgga 60ggccaggcca gcctgacctg cctggtgaaa gactacttcc ccgagcccgt gaccgtgtcc 120tggaactctg gcgccctgac cagcggcgtg aaaaccaccc ctgctgtgct ggacagcagc 180ggcctgtact tcctgtacag caaagtgacc gtgcctagca gcagcctggg cacccagacc 240tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa agtg 29455321DNAArtificial SequenceSynthetic polynucleotide 55agaaccgtgg ctgctcctca ggtgtacatc ctgcctccca gcgacgatga gctgaagtct 60ggcaccgcca gcgtgacatg tctgctgaag aacttctacc cccgcgaggc caaggtgcag 120tggaaagtgg acaacgccct gcagagcggc aacagcaaag aaaccgtggt ggaacaggac 180agcaaggact ccacctactt tctgtactcc aagctgaccc tgagcaaggc cgactacgag 240aagcacaagg tgtacgcctg cgaagtgacc caccagggcc tgagcagccc cgtgaccaag 300agcttcaacc ggggcgagtg c 321561347DNAArtificial SequenceSynthetic polynucleotide 56caggtccagc tgaagcagag cggcccaggc ctcgtgcagc cctctcagtc tctcagcatc 60acctgcaccg tgtccggctt cagcctgacc aactacggcg tgcactgggt ccgccagagc 120cccggcaagg gcctggaatg gctgggcgtg atctggtccg gcggcaacac cgactacaac 180acccccttca ccagcaggct gtccatcaac aaggacaaca gcaagagcca ggtgttcttc 240aagatgaaca gcctgcagag caacgacacc gccatctact actgcgccag ggctctgacc 300tactacgact acgagttcgc ctactgggga cagggcaccc tggtcactgt ctccgccgcc 360agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac ctctggcggc 420acagccgccc tgggctgcct ggtcaaggac tacttccccg agcccgtgac agtgtcctgg 480aacagcggag ccctgacctc cggcgtccac accttccccg ccgtgctgca gagcagcggc 540ctgtacagcc tgagcagcgt ggtcacagtg ccctctagca gcctcggcac ccagacctac 600atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagaaggt ggagcccaag 660agctgcgaca aaactcacac atgccccccc tgccctgccc ctgagctgct gggcggaccc 720tccgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 780gtgacctgcg tggtggtgga cgtgagccac gaggaccctg aggtgaagtt caattggtac 840gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggaaca gtacaacagc 900acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 960tacaagtgca aggtctccaa caaggccctg cctgccccca tcgagaaaac catctccaaa 1020gccaaaggcc agcccagaga accccaggtg tacaccctgc cccccagcag agatgagctg 1080accaagaacc aggtcaagct cgtgtgcctg gtcaccggct tctaccccag cgatatcgcc 1140gtggagtggg agagcaacgg ccagcctgaa aacaactact acaccacccc ccctgtgctg 1200gacagcgacg gcagcttcag cctggtgtcc tggctgaacg tggacaagag ccggtggcag 1260cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320aagtccctga gcctgtctcc tggcaag 134757642DNAArtificial SequenceSynthetic polynucleotide 57gacatcctgc tgacccagag ccccgtgatc ctgagcgtgt ccccaggcga gagggtgtcc 60ttcagctgca gagccagcca gagcatcggc accaacatcc actggtatca gcagaggacc 120aacggcagcc ccaggctgct gatcaagtac gccagcgagt ccatcagcgg catccccagc 180aggttcagcg gcagcggctc cggcaccgac ttcaccctga gcatcaacag cgtggagagc 240gaggacatcg ccgactacta ctgccagcag aacaacaact ggcccaccac cttcggagcc 300ggcaccaagc tggaactgaa gaggaccgtg gctgccccca gcgtgttcat cttccccccc 360agcgacgagc agctgaagtc cggcaccgcc agcgtggtct gcctgctgaa caacttctac 420ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480gaaagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtcac ccaccagggc 600ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642581422DNAArtificial SequenceSynthetic polynucleotide 58gaggtgcagc tggtcgagtc tggcggcgga ctggtgcagc ctggcggctc cctgcggctg 60tcctgcgccg cctccggctt caacatcaag gacacctaca tccactgggt gcggcaggcc 120cctggcaagg gcctggagtg ggtggcccgg atctacccta ccaacggcta caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga 300ggagatggct tctacgccat ggactactgg ggccagggca ccctggtgac cgtgtcctcc 360ggtggcggtg gcagcggcgg tggtggttcc ggaggcggcg gttctgacat ccagatgacc 420cagtccccct ccagcctgtc tgcctccgtg ggcgaccggg tgaccatcac ctgccgggcc 480tcccaggacg tgaacaccgc cgtggcctgg tatcagcaga agcctggcaa ggcccctaag 540ctgctgatct actccgcctc cttcctgtac tccggcgtgc cttcccggtt ctccggctcc 600cggtccggca ccgacttcac cctgaccatc tcctccctgc agcctgagga cttcgccacc 660tactactgcc agcagcacta caccacccct cctaccttcg gccagggcac caaggtggag 720atcaagggag gaggagggtc agacaaaact cacacttgcc caccgtgccc agcacctgaa 780ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840tcccggaccc ctgaggtcac ctgcgtggtg gtggacgtga gccacgagga ccctgaggtg 900aagttcaatt ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gccccgggag 960gaacagtaca acagcaccta ccgggtggtg tccgtgctga ccgtgctgca ccaggactgg 1020ctgaacggca aggaatacaa gtgcaaggtc tccaacaagg ccctgcctgc ccccatcgaa 1080aagaccatca gcaaggccaa gggacagccc agggaacctg aggtggccac attcccacct 1140agccgggacg agctgacaaa aaatcaggtc accctcgtct gtctcgtgac cggcttttac 1200ccttccgaca ttgccgtgga atgggaatcc aatgggcagc ccgagaacaa ttacaagaca 1260gacccccccc tgctggaatc cgatggcagc ttctgcctga gcagccggct gcgggtggac 1320aagtccagat ggcagcaggg gaatgtcttt tcctgctccg tcatgcatga agccctccac 1380aatcattata cacagaaaag cctgagcctg agccccggca ag 142259449PRTArtificial SequenceSynthetic polypeptide 59Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Lys Leu Val 355 360 365Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys60214PRTArtificial SequenceSynthetic polypeptide 60Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21061474PRTArtificial SequenceSynthetic polypeptide 61Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala145 150 155 160Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly 180 185 190Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu 195 200 205Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu225 230 235 240Ile Lys Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260

265 270Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu305 310 315 320Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp Glu 370 375 380Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe Tyr385 390 395 400Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly Ser Phe Ala 420 425 430Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys465 47062851PRTArtificial SequenceSynthetic polypeptide 62Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln1 5 10 15Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn 20 25 30Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg 35 40 45Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr 50 55 60Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu65 70 75 80Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala 85 90 95Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro 100 105 110Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn 115 120 125Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val 130 135 140Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu145 150 155 160Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp 165 170 175Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala 180 185 190Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200 205His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys 210 215 220Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys225 230 235 240Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn 245 250 255Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro 260 265 270Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly 275 280 285Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys 290 295 300Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu305 310 315 320Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys 325 330 335Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe 340 345 350Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu 355 360 365Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln 370 375 380Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu385 390 395 400Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val 405 410 415Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 420 425 430Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440 445Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr 450 455 460Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln465 470 475 480Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro 485 490 495Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val 500 505 510Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn 515 520 525Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn 530 535 540Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His545 550 555 560Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met 565 570 575Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 580 585 590Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly 595 600 605Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Gly Gly Gly 610 615 620Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly625 630 635 640Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 645 650 655Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 660 665 670Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 675 680 685His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 690 695 700Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly705 710 715 720Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 725 730 735Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 740 745 750Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 755 760 765Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 770 775 780Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro785 790 795 800Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 805 810 815Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 820 825 830His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 835 840 845Pro Gly Lys 85063862PRTArtificial SequenceSynthetic polypeptide 63Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser1 5 10 15Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val65 70 75 80Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr145 150 155 160Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195 200 205Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly 210 215 220Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly225 230 235 240Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys305 310 315 320Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser 325 330 335Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu 340 345 350Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu385 390 395 400Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420 425 430Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435 440 445Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe 450 455 460Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp465 470 475 480Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp545 550 555 560Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala 565 570 575Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620Arg Ala Ser Pro Leu Thr Glu Pro Lys Ser Cys Asp Lys Thr His Thr625 630 635 640Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 645 650 655Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 660 665 670Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 675 680 685Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 690 695 700Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val705 710 715 720Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 725 730 735Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 740 745 750Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 755 760 765Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 770 775 780Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly785 790 795 800Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 805 810 815Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 820 825 830Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 835 840 845Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 850 855 860642577DNAArtificial SequenceSynthetic polynucleotide 64ctggaggaaa agaaagtttg ccaaggcacg agtaacaagc tcacgcagtt gggcactttt 60gaagatcatt ttctcagcct ccagaggatg ttcaataact gtgaggtggt ccttgggaat 120ttggaaatta cctatgtgca gaggaattat gatctttcct tcttaaagac catccaggag 180gtggctggtt atgtcctcat tgccctcaac acagtggagc gaattccttt ggaaaacctg 240cagatcatca gaggaaatat gtactacgaa aattcctatg ccttagcagt cttatctaac 300tatgatgcaa ataaaaccgg actgaaggag ctgcccatga gaaatttaca ggaaatcctg 360catggcgccg tgcggttcag caacaaccct gccctgtgca acgtggagag catccagtgg 420cgggacatag tcagcagtga ctttctcagc aacatgtcga tggacttcca gaaccacctg 480ggcagctgcc aaaagtgtga tccaagctgt cccaatggga gctgctgggg tgcaggagag 540gagaactgcc agaaactgac caaaatcatc tgtgcccagc agtgctccgg gcgctgccgt 600ggcaagtccc ccagtgactg ctgccacaac cagtgtgctg caggctgcac aggcccccgg 660gagagcgact gcctggtctg ccgcaaattc cgagacgaag ccacgtgcaa ggacacctgc 720cccccactca tgctctacaa ccccaccacg taccagatgg atgtgaaccc cgagggcaaa 780tacagctttg gtgccacctg cgtgaagaag tgtccccgta attatgtggt gacagatcac 840ggctcgtgcg tccgagcctg tggggccgac agctatgaga tggaggaaga cggcgtccgc 900aagtgtaaga agtgcgaagg gccttgccgc aaagtgtgta acggaatagg tattggtgaa 960tttaaagact cactctccat aaatgctacg aatattaaac acttcaaaaa ctgcacctcc 1020atcagtggcg atctccacat cctgccggtg gcatttaggg gtgactcctt cacacatact 1080cctcctctgg atccacagga actggatatt ctgaaaaccg taaaggaaat cacagggttt 1140ttgctgattc aggcttggcc tgaaaacagg acggacctcc atgcctttga gaacctagaa 1200atcatacgcg gcaggaccaa gcaacatggt cagttttctc ttgcagtcgt cagcctgaac 1260ataacatcct tgggattacg ctccctcaag gagataagtg atggagatgt gataatttca 1320ggaaacaaaa atttgtgcta tgcaaataca ataaactgga aaaaactgtt tgggacctcc 1380ggtcagaaaa ccaaaattat aagcaacaga ggtgaaaaca gctgcaaggc cacaggccag 1440gtctgccatg ccttgtgctc ccccgagggc tgctggggcc cggagcccag ggactgcgtc 1500tcttgccgga atgtcagccg aggcagggaa tgcgtggaca agtgcaacct tctggagggt 1560gagccaaggg agtttgtgga gaactctgag tgcatacagt gccacccaga gtgcctgcct 1620caggccatga acatcacctg cacaggacgg ggaccagaca actgtatcca gtgtgcccac 1680tacattgacg gcccccactg cgtcaagacc tgcccggcag gagtcatggg agaaaacaac 1740accctggtct ggaagtacgc agacgccggc catgtgtgcc acctgtgcca tccaaactgc 1800acctacggat gcactgggcc aggtcttgaa ggctgtccaa cgaatgggcc taagatcccg 1860tccggtggtg gtggtggtac ccacacctgc cccccctgcc ctgcccctga gctgctgggc 1920ggacccagcg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagccggacc 1980cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accctgaggt gaagttcaat 2040tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac 2100aactccacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 2160aaggaataca agtgcaaggt gtccaacaag gccctgcctg cccccatcga aaagaccatc 2220agcaaggcca agggccagcc cagggagccc caggtgtaca ccctgccccc cagccgggag 2280gagatgacca agaaccaggt gtccctgacc tgcctggtga agggcttcta ccccagcgac 2340atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct 2400gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagagcagg 2460tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 2520acccagaaga gcctgagcct gtcccccggc aagagcgccc atcatcacca tcaccat 2577652586DNAArtificial SequenceSynthetic polynucleotide 65acccaagtgt gcaccggcac agacatgaag ctgcggctcc ctgccagtcc cgagacccac 60ctggacatgc tccgccacct ctaccagggc tgccaggtgg tgcagggaaa cctggaactc 120acctacctgc ccaccaatgc cagcctgtcc ttcctgcagg atatccagga ggtgcagggc 180tacgtgctca tcgctcacaa ccaagtgagg caggtcccac tgcagaggct gcggattgtg 240cgaggcaccc agctctttga ggacaactat gccctggccg tgctagacaa tggagacccg 300ctgaacaata ccacccctgt cacaggggcc tccccaggag gcctgcggga gctgcagctt 360cgaagcctca cagagatctt gaaaggaggg gtcttgatcc agcggaaccc ccagctctgc 420taccaggaca cgattttgtg gaaggacatc ttccacaaga acaaccagct ggctctcaca 480ctgatagaca ccaaccgctc tcgggcctgc cacccctgtt ctccgatgtg taagggctcc 540cgctgctggg gagagagttc tgaggattgt cagagcctga cgcgcactgt ctgtgccggt 600ggctgtgccc

gctgcaaggg gccactgccc actgactgct gccatgagca gtgtgctgcc 660ggctgcacgg gccccaagca ctctgactgc ctggcctgcc tccacttcaa ccacagtggc 720atctgtgagc tgcactgccc agccctggtc acctacaaca cagacacgtt tgagtccatg 780cccaatcccg agggccggta tacattcggc gccagctgtg tgactgcctg tccctacaac 840tacctttcta cggacgtggg atcctgcacc ctcgtctgcc ccctgcacaa ccaagaggtg 900acagcagagg atggaacaca gcggtgtgag aagtgcagca agccctgtgc ccgagtgtgc 960tatggtctgg gcatggagca cttgcgagag gtgagggcag ttaccagtgc caatatccag 1020gagtttgctg gctgcaagaa gatctttggg agcctggcat ttctgccgga gagctttgat 1080ggggacccag cctccaacac tgccccgctc cagccagagc agctccaagt gtttgagact 1140ctggaagaga tcacaggtta cctatacatc tcagcatggc cggacagcct gcctgacctc 1200agcgtcttcc agaacctgca agtaatccgg ggacgaattc tgcacaatgg cgcctactcg 1260ctgaccctgc aagggctggg catcagctgg ctggggctgc gctcactgag ggaactgggc 1320agtggactgg ccctcatcca ccataacacc cacctctgct tcgtgcacac ggtgccctgg 1380gaccagctct ttcggaaccc gcaccaagct ctgctccaca ctgccaaccg gccagaggac 1440gagtgtgtgg gcgagggcct ggcctgccac cagctgtgcg cccgagggca ctgctggggt 1500ccagggccca cccagtgtgt caactgcagc cagttccttc ggggccagga gtgcgtggag 1560gaatgccgag tactgcaggg gctccccagg gagtatgtga atgccaggca ctgtttgccg 1620tgccaccctg agtgtcagcc ccagaatggc tcagtgacct gttttggacc ggaggctgac 1680cagtgtgtgg cctgtgccca ctataaggac cctcccttct gcgtggcccg ctgccccagc 1740ggtgtgaaac ctgacctctc ctacatgccc atctggaagt ttccagatga ggagggcgca 1800tgccagcctt gccccatcaa ctgcacccac tcctgtgtgg acctggatga caagggctgc 1860cccgccgagc agagagccag ccctctgacg gagcccaaga gctgcgacaa gacccacacc 1920tgccccccct gccctgcccc tgagctgctg ggcggaccca gcgtgttcct gttccccccc 1980aagcccaagg acaccctgat gatcagccgg acccccgagg tgacctgcgt ggtggtggac 2040gtgagccacg aggaccctga ggtgaagttc aattggtacg tggacggcgt ggaggtgcac 2100aacgccaaga ccaagccccg ggaggagcag tacaactcca cctaccgggt ggtgtccgtg 2160ctgaccgtgc tgcaccagga ctggctgaac ggcaaggaat acaagtgcaa ggtgtccaac 2220aaggccctgc ctgcccccat cgaaaagacc atcagcaagg ccaagggcca gcccagggag 2280ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgtccctg 2340acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 2400cagcccgaga acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc 2460ctgtacagca agctgaccgt ggacaagagc aggtggcagc agggcaacgt gttcagctgc 2520agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgtccccc 2580ggcaag 2586661365DNAArtificial SequenceSynthetic polynucleotide 66caggtgcagt tgcaggagag cggcccaggc ctggtgaagc ccagcgagac actgagcctg 60acctgcaccg tgagcggctt cagcctgacc aactacggca tccactggat caggcagccc 120ccaggcaagg gcctggagtg gctgggcgtg atctgggcca ggggcttcac caactacaac 180agcgccctga tgagcaggct gaccatcagc aaggacaaca gcaagaacca ggtgtccctg 240aagctgtcca gcgtgacagc cgccgacacc gccgtgtact actgcgccag ggccaacgac 300ggcgtgtact acgccatgga ctactggggc cagggcaccc tggtcaccgt cagctcagcg 360tcgaccaagg gccccagcgt gttcccgcta gcccccagca gcaagagcac cagcggcggc 420acagccgccc tgggctgcct ggtgaaggac tacttccccg agcccgttac cgtgtcctgg 480aactctggag ccctgacctc cggcgtgcac accttccccg ccgtgctcca gagcagcggc 540ctgtacagcc tgagcagcgt ggtgacagtg cccagcagca gcctgggaac ccagacctac 600atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagaaggt ggagcccaag 660agctgcgaca aaactcacac atgccccccc tgccctgccc ctgagctgct gggcggaccc 720tccgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 780gtgacctgcg tggtggtgga cgtgagccac gaggaccctg aggtgaagtt caattggtac 840gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggaaca gtacaacagc 900acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 960tacaagtgca aggtctccaa caaggccctg cctgccccca tcgagaaaac catctccaaa 1020gccaaaggcc agcccagaga accccaggtg tacaccctgc cccccagcag agatgagctg 1080accaagaacc aggtcaagct cgtgtgcctg gtcaccggct tctaccccag cgatatcgcc 1140gtggagtggg agagcaacgg ccagcctgaa aacaactact acaccacccc ccctgtgctg 1200gacagcgacg gcagcttcag cctggtgtcc tggctgaacg tggacaagag ccggtggcag 1260cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320aagtccctga gcctgtctcc tggcaagcat caccatcacc atcac 136567639DNAArtificial SequenceSynthetic polynucleotide 67gacttcgtga tgacccagag ccccgccttc ctgagcgtga ccccaggcga gaaggtgacc 60atcacctgca gcgcccagag cagcgtgaac tacatccact ggtaccagca gaagcccgac 120caggccccca agaagctgat ctacgacacc agcaagctgg ccagcggcgt gcccagcagg 180ttcagcggca gcggctccgg caccgactac accttcacca tcagcagcct ggaggccgag 240gacgccgcca cctactactg ccagcagtgg accaccaacc ccctgacctt cggccagggc 300accaaggtgg agatcaagag gaccgtggcc gcccccagcg tgttcatctt cccccccagc 360gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420cgggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 540agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600tccagccccg tgaccaagag cttcaacagg ggcgagtgc 63968455PRTArtificial SequenceSynthetic polypeptide 68Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Lys Leu Val 355 360 365Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys His His His His His His 450 45569213PRTArtificial SequenceSynthetic polypeptide 69Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 21070450PRTArtificial SequenceSynthetic polypeptide 70Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Met Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Asn 130 135 140Leu Ala Cys Leu Val Lys Glu Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val Phe Thr Ala Pro Ala Val 165 170 175Leu Ser Ser Ser Gly Leu Tyr Asn Leu Val Ser Leu Val Lys Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala 340 345 350Thr Phe Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu 355 360 365Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu385 390 395 400Leu Glu Ser Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys 45071445PRTArtificial SequenceSynthetic polypeptide 71Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Gln Val 115 120 125Tyr Pro Leu Ala Pro Ser Ser Asp Glu Thr Ser Gly Gly Gln Ala Ser 130 135 140Leu Thr Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val Lys Thr Thr Pro Ala Val 165 170 175Leu Asp Ser Ser Gly Leu Tyr Phe Leu Tyr Ser Lys Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly385 390 395 400Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 44572449PRTArtificial SequenceSynthetic polypeptide 72Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185

190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Lys Leu Val 355 360 365Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys73227PRTArtificial SequenceSynthetic polypeptide 73Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Lys 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Tyr Thr Thr Pro Pro 165 170 175Met Leu Asp Ser Asp Gly Ser Phe Ser Leu Val Ser Trp Leu Asn Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22574227PRTArtificial SequenceSynthetic polypeptide 74Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125Ala Thr Phe Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr 130 135 140Leu Val Cys Leu Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro 165 170 175Leu Leu Glu Ser Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22575335PRTArtificial SequenceSynthetic polypeptide 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu 245 250 255Val Thr Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser 275 280 285Asp Gly Ser Phe Ala Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33576107PRTArtificial SequenceSynthetic polypeptide 76Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu1 5 10 15Glu Met Thr Lys Asn Gln Val Lys Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu 35 40 45Asn Asn Tyr Tyr Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 50 55 60Ser Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10577321DNAArtificial SequenceSynthetic polynucleotide 77ggccagccca gagaacccca ggtgtacacc ctgcccccca gccgggaaga gatgaccaag 60aaccaggtga aactggtgtg cctggtgaca ggcttctacc ccagcgatat cgccgtggaa 120tgggagagca gcggccagcc tgagaacaac tactacacca ccccccccat gctggacagc 180gacggcagct tcagcctggt gtcctggctg aacgtggaca agagccggtg gcagcagggc 240aacatcttca gctgcagcgt gatgcacgag gccctgcaca accggttcac ccagaagtcc 300ctgagcctga gccccggcaa g 32178107PRTArtificial SequenceSynthetic polypeptide 78Gly Gln Pro Arg Glu Pro Glu Val His Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Arg Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro Pro Arg Leu Glu Ser Asp Gly Ser Phe 50 55 60Ala Leu Thr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10579107PRTArtificial SequenceSynthetic polypeptide 79Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Thr Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Leu Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Ile Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 10580227PRTArtificial SequenceSynthetic polypeptide 80Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125His Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Arg Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Arg Leu Glu Ser Asp Gly Ser Phe Ala Leu Thr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22581335PRTArtificial SequenceSynthetic polypeptide 81Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235 240Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Thr Leu Thr Cys Leu 245 250 255Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly Gln Pro Glu Asn Asn Tyr Leu Thr Trp Pro Pro Val Leu Asp Ser 275 280 285Asp Gly Ser Phe Phe Leu Tyr Ser Ile Leu Arg Val Asp Lys Ser Arg 290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 33582321DNAArtificial SequenceSynthetic polynucleotide 82ggccagccta gagaacctga ggtgcacacc ctgcccccca gcagagatga gctgaccaag 60aaccaggtgt ccctgacctg tctcgtgcgg ggcttctacc cctccgatat cgccgtggaa 120tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccag actggaaagc 180gacggcagct ttgccctgac cagcaagctg accgtggaca agagcagatg gcagcagggc 240aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 300ctgagcctga gccccggcaa g 32183321DNAArtificial SequenceSynthetic polynucleotide 83ggccagccta gagaacccca ggtgtacaca ctgcccccca gcagagatga gctgaccaag 60aaccaagtga ccctgacctg cctcgtgaag ggcttctacc cctccgatat cgccgtggaa 120tgggagagca acggccagcc cgagaacaac tacctgacct ggccccctgt gctggacagc 180gacggctcat tcttcctgta cagcatcctg cgggtggaca agagcagatg gcagcagggc 240aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 300ctgagcctga gccccggcaa g 32184113PRTArtificial SequenceSynthetic polypeptide 84Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg1 5 10 15Glu Gln Leu Asn Leu Arg Glu Ser Ala Lys Ile Val Cys Leu Val Thr 20 25 30Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln 35 40 45Pro Leu Ser Pro Glu Lys Tyr Tyr Thr Ser Ala Pro Met Pro Glu Pro 50 55 60Gln Ala Pro Gly Arg Tyr Ser Ala Val Ser Trp Leu Asn Val Ser Glu65 70 75 80Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu 85 90 95Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly 100 105 110Lys85113PRTArtificial SequenceSynthetic polypeptide 85Gly Val Ala Leu His Arg Pro Glu Val Ala Leu Phe Pro Pro Ala Arg1 5 10 15Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Val Cys Leu Val Thr 20 25 30Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln 35 40 45Pro Leu Ser Pro Glu Lys Tyr Val Thr Asp Ala Pro Leu Pro Glu Pro 50 55 60Gln Ala Pro Gly Arg Tyr Ala Ala Ser Ser Arg Leu Arg Val Ser Glu65 70 75 80Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu 85 90 95Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly 100 105 110Lys86339DNAArtificial SequenceSynthetic polynucleotide 86ggcgtggccc tgcacagacc cgacgtgtac ctgctgcctc ctgccagaga gcagctgaac 60ctgcgggaaa gcgccaagat cgtgtgtctc gtgaccggct tctcccctgc cgacgtgttc 120gtgcagtgga tgcagagagg ccagcccctg agccccgaga agtactacac aagcgccccc 180atgcctgagc cacaggcccc tggaagatac agcgccgtgt cttggctgaa cgtgtccgag 240gaagagtgga acaccggcga gacatacacc tgtgtggtgg cccatgaggc cctgcccaat 300agagtgaccg

agcggaccgt ggacaagagc accggaaag 33987339DNAArtificial SequenceSynthetic polynucleotide 87ggcgtggccc tgcacagacc agaggtggcc ctgtttccac ccgccagaga gcagctgaac 60ctgcgggaaa gcgccaccat cgtgtgtctc gtgaccggct tcagccctgc cgacgtgttc 120gtgcagtgga tgcagagagg ccagcccctg tcccccgaga aatacgtgac agacgccccc 180ctgcctgagc cacaggcccc tggaagatat gccgccagca gcagactgcg ggtgtccgag 240gaagagtgga acaccggcga gacatacacc tgtgtggtgg cccatgaggc cctgcccaat 300agagtgaccg aacggaccgt ggacaagagc accggcaag 33988239PRTArtificial SequenceSynthetic polypeptide 88Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Val Ala Leu His Arg Pro Asp 115 120 125Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser 130 135 140Ala Lys Ile Val Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val Phe145 150 155 160Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Tyr 165 170 175Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr Ser Ala 180 185 190Val Ser Trp Leu Asn Val Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr 195 200 205Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val Thr Glu 210 215 220Arg Thr Val Asp Lys Ser Thr Gly Lys His His His His His His225 230 23589341PRTArtificial SequenceSynthetic polypeptide 89Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Val Ala Leu His Arg Pro Glu Val Ala Leu Phe225 230 235 240Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Val 245 250 255Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met 260 265 270Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Asp Ala Pro 275 280 285Leu Pro Glu Pro Gln Ala Pro Gly Arg Tyr Ala Ala Ser Ser Arg Leu 290 295 300Arg Val Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val305 310 315 320Val Ala His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp 325 330 335Lys Ser Thr Gly Lys 34090110PRTArtificial SequenceSynthetic polypeptide 90Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp1 5 10 15Pro Gly Ser Arg Asp Lys Arg Lys Leu Val Cys Leu Ile Thr Asn Phe 20 25 30Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu 35 40 45Pro Asp Ala Arg His Tyr Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser 50 55 60Gly Phe Ser Val Val Ser Trp Leu Asn Val Thr Arg Ala Glu Trp Glu65 70 75 80Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro 85 90 95Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 100 105 11091110PRTArtificial SequenceSynthetic polypeptide 91Gly Pro Arg Ala Ala Pro Glu Val Ala Ala Phe Ala Thr Pro Glu Trp1 5 10 15Pro Gly Ser Arg Asp Lys Arg Thr Leu Val Cys Leu Ile Thr Asn Phe 20 25 30Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu 35 40 45Pro Asp Ala Arg His Ser Thr Asp Gln Pro Leu Lys Glu Lys Gly Ser 50 55 60Gly Phe Ala Val Ser Ser Leu Leu Arg Val Thr Arg Ala Glu Trp Glu65 70 75 80Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro 85 90 95Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 100 105 11092330DNAArtificial SequenceSynthetic polynucleotide 92ggacctagag ccgcccctga ggtgtacgcc tttgccacac ctgagtggcc cggcagccgg 60gacaagagaa aactcgtgtg cctgatcacc aacttcatgc ccgaggacat cagcgtgcag 120tggctgcaca acgaggtgca gctgcccgac gccagacact acaccaccca gcccagaaag 180accaagggca gcggcttcag cgtggtgtcc tggctgaatg tgaccagagc cgagtgggag 240cagaaggacg agttcatctg cagagccgtg cacgaggccg ccagcccttc tcagacagtg 300cagagggccg tgtccgtgaa ccctggaaaa 33093330DNAArtificial SequenceSynthetic polynucleotide 93ggacctagag ccgctcctga agtggccgcc tttgccacac ctgagtggcc cggcagcaga 60gacaagagaa ccctcgtgtg cctgatcacc aacttcatgc ccgaggacat cagcgtgcag 120tggctgcaca acgaggtgca gctgcccgat gccagacaca gcaccgacca gcccctgaaa 180gagaagggca gcggctttgc cgtgtccagc ctgctgagag tgaccagagc cgagtgggag 240cagaaggacg agttcatctg cagagccgtg cacgaggccg ccagcccttc tcagacagtg 300cagagggccg tgtccgtgaa ccctggaaaa 33094236PRTArtificial SequenceSynthetic polypeptide 94Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Pro Arg Ala Ala Pro Glu Val 115 120 125Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Lys 130 135 140Leu Val Cys Leu Ile Thr Asn Phe Met Pro Glu Asp Ile Ser Val Gln145 150 155 160Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Tyr Thr Thr 165 170 175Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Ser Val Val Ser Trp Leu 180 185 190Asn Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg 195 200 205Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val 210 215 220Ser Val Asn Pro Gly Lys His His His His His His225 230 23595338PRTArtificial SequenceSynthetic polypeptide 95Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Asp Lys Thr His 100 105 110Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala Lys Gly Pro Arg Ala Ala Pro Glu Val Ala Ala Phe Ala225 230 235 240Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Val Cys Leu 245 250 255Ile Thr Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn 260 265 270Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Asp Gln Pro Leu Lys 275 280 285Glu Lys Gly Ser Gly Phe Ala Val Ser Ser Leu Leu Arg Val Thr Arg 290 295 300Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu305 310 315 320Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro 325 330 335Gly Lys96111PRTHomo sapiensMISC_FEATURE(1)..(111)human CH3 (isotype alpha 1) constant domain 96Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu1 5 10 15Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly 20 25 30Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu 35 40 45Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser 50 55 60Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala65 70 75 80Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu 85 90 95Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala 100 105 11097111PRTHomo sapiensMISC_FEATURE(1)..(111)human CH3 (isotype alpha 2) constant domain 97Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu1 5 10 15Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly 20 25 30Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu 35 40 45Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser 50 55 60Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala65 70 75 80Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu 85 90 95Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala 100 105 11098353PRTArtificial SequenceIGHA1 98Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys Ser Thr1 5 10 15Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val 35 40 45Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly65 70 75 80Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115 120 125Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn 130 135 140Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe145 150 155 160Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu 165 170 175Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys 180 185 190Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr 195 200 205Pro Glu Ser Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn 210 215 220Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu225 230 235 240Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser 245 250 255Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro 260 265 270Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly 275 280 285Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp 290 295 300Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu305 310 315 320Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro 325 330 335Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys 340 345 350Tyr99340PRTArtificial SequenceIGHA2 99Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr1 5 10 15Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly65 70 75 80Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val Pro Cys Pro Val Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly145 150 155 160Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180 185 190Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys 195 200 205Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser 210 215 220Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr

Cys Leu Ala Arg225 230 235 240Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265 270Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His 290 295 300Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala305 310 315 320Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp 325 330 335Gly Thr Cys Tyr 340100384PRTArtificial SequenceIGHD 100Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg1 5 10 15His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25 30Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser 35 40 45Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr 50 55 60Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly65 70 75 80Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu 85 90 95Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro 100 105 110Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala 115 120 125Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135 140Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu145 150 155 160Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala 165 170 175Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val 180 185 190Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly 195 200 205Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser 210 215 220Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu225 230 235 240Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu 245 250 255Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro 260 265 270Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala 275 280 285Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295 300Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe305 310 315 320Ala Pro Ala Arg Pro Pro Pro Gln Pro Arg Ser Thr Thr Phe Trp Ala 325 330 335Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr 340 345 350Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala 355 360 365Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met Lys 370 375 380101428PRTArtificial SequenceIGHE 101Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys1 5 10 15Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr 20 25 30Gly Tyr Phe Pro Glu Pro Val Met Val Thr Cys Asp Thr Gly Ser Leu 35 40 45Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser Gly 50 55 60His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala Trp Ala Lys65 70 75 80Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser Ser Thr Asp Trp 85 90 95Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr Pro Pro 100 105 110Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro 115 120 125Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr 130 135 140Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu145 150 155 160Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser 165 170 175Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr 180 185 190Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys 195 200 205Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro 210 215 220Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu225 230 235 240Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser 245 250 255Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys 260 265 270Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr 275 280 285Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro 290 295 300His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro305 310 315 320Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly 325 330 335Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro 340 345 350Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp 355 360 365Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe 370 375 380Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys385 390 395 400Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln 405 410 415Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 420 425102330PRTArtificial SequenceIGHG1 102Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330103326PRTArtificial SequenceIGHG2 103Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly Lys 325104377PRTArtificial SequenceIGHG3 104Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro145 150 155 160Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His225 230 235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn305 310 315 320Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375105327PRTArtificial SequenceIGHG4 105Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly Lys 325106431PRTArtificial SequenceIGHGP 106Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Val Pro Ser Ser Arg1 5 10 15Ser Val Ser Glu Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Arg 35 40 45Ser Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Pro Lys Thr Pro Cys Cys Asp Thr Thr His Thr Cys Pro Pro 100 105 110Cys Ala Thr Glu Pro Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

Lys Pro Trp Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr His Val Val Ser Val Leu Thr Val Val 180 185 190His Gln Asn Trp Leu Asn Gly Arg Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gln 210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Lys Met Thr225 230 235 240Lys Asn Gln Val Thr Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 245 250 255Asp Ile Thr Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 260 265 270Lys Thr Thr Pro Pro Met Leu Asp Ser Asn Gly Ser Phe Phe Leu Tyr 275 280 285Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 290 295 300Ser Cys Ser Val Met His Glu Gly Leu His Asn His Tyr Thr Gln Lys305 310 315 320Ser Leu Ser Leu Ser Pro Gly Lys Gln Pro Arg Glu Pro Gln Val Tyr 325 330 335Thr Leu Pro Pro Ser Gln Lys Met Thr Lys Asn Gln Val Thr Leu Thr 340 345 350Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Thr Val Glu Trp Glu 355 360 365Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 370 375 380Asp Ser Asn Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys385 390 395 400Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 405 410 415Gly Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 420 425 430107452PRTArtificial SequenceIGHM 107Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn1 5 10 15Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30Phe Leu Pro Asp Ser Ile Thr Leu Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln65 70 75 80Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr145 150 155 160Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln 180 185 190Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr225 230 235 240Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Ser Glu Glu Glu 290 295 300Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Ala His Glu Ala Leu Pro305 310 315 320Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr 325 330 335Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 340 345 350Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln 355 360 365Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr 370 375 380Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr385 390 395 400Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln 405 410 415Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser 420 425 430Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser 435 440 445Ile Leu Thr Val 450108107PRTArtificial SequenceIGKC 108Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105109104PRTArtificial SequenceIGLC1 109Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu1 5 10 15Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 20 25 30Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 35 40 45Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 50 55 60Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His65 70 75 80Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 85 90 95Thr Val Ala Pro Thr Glu Cys Ser 100110105PRTArtificial SequenceIGLC2 110Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105111104PRTArtificial SequenceIGLC3 111Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu1 5 10 15Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 20 25 30Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 35 40 45Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 50 55 60Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His65 70 75 80Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 85 90 95Thr Val Ala Pro Thr Glu Cys Ser 100112105PRTArtificial SequenceIGLC6 112Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Lys Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45Val Asn Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr Val Ala Pro Ala Glu Cys Ser 100 105113106PRTArtificial SequenceIGLC7 113Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45Ala Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105114141PRTArtificial SequenceTRACmisc_feature(1)..(1)Xaa can be any naturally occurring amino acid 114Xaa Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys1 5 10 15Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr 20 25 30Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 35 40 45Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala 50 55 60Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser65 70 75 80Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp 85 90 95Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe 100 105 110Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala 115 120 125Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140115177PRTArtificial SequenceTRBC1 115Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu65 70 75 80Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90 95Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 100 105 110Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 115 120 125Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser 130 135 140Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala145 150 155 160Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp 165 170 175Phe116178PRTArtificial SequenceTRBC2 116Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser1 5 10 15Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 20 25 30Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 35 40 45Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu 50 55 60Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg65 70 75 80Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 85 90 95Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 100 105 110Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 115 120 125Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala 130 135 140Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val145 150 155 160Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser 165 170 175Arg Gly117154PRTArtificial SequenceTRDCmisc_feature(1)..(1)Xaa can be any naturally occurring amino acid 117Xaa Ser Gln Pro His Thr Lys Pro Ser Val Phe Val Met Lys Asn Gly1 5 10 15Thr Asn Val Ala Cys Leu Val Lys Glu Phe Tyr Pro Lys Asp Ile Arg 20 25 30Ile Asn Leu Val Ser Ser Lys Lys Ile Thr Glu Phe Asp Pro Ala Ile 35 40 45Val Ile Ser Pro Ser Gly Lys Tyr Asn Ala Val Lys Leu Gly Lys Tyr 50 55 60Glu Asp Ser Asn Ser Val Thr Cys Ser Val Gln His Asp Asn Lys Thr65 70 75 80Val His Ser Thr Asp Phe Glu Val Lys Thr Asp Ser Thr Asp His Val 85 90 95Lys Pro Lys Glu Thr Glu Asn Thr Lys Gln Pro Ser Lys Ser Cys His 100 105 110Lys Pro Lys Ala Ile Val His Thr Glu Lys Val Asn Met Met Ser Leu 115 120 125Thr Val Leu Gly Leu Arg Met Leu Phe Ala Lys Thr Val Ala Val Asn 130 135 140Phe Leu Leu Thr Ala Lys Leu Phe Phe Leu145 150118173PRTArtificial SequenceTRGC1 118Asp Lys Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu1 5 10 15Pro Ser Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys 20 25 30Leu Leu Glu Lys Phe Phe Pro Asp Val Ile Lys Ile His Trp Gln Glu 35 40 45Lys Lys Ser Asn Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met Lys 50 55 60Thr Asn Asp Thr Tyr Met Lys Phe Ser Trp Leu Thr Val Pro Glu Lys65 70 75 80Ser Leu Asp Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn Lys 85 90 95Asn Gly Val Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val 100 105 110Ile Thr Met Asp Pro Lys Asp Asn Cys Ser Lys Asp Ala Asn Asp Thr 115 120 125Leu Leu Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr Leu Leu 130 135 140Leu Leu Leu Lys Ser Val Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu145 150 155 160Leu Arg Arg Thr Ala Phe Cys Cys Asn Gly Glu Lys Ser 165 170119109PRTArtificial SequenceTRGC2 (2X) 119Lys Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu Pro1 5 10 15Ser Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu 20 25 30Leu Glu Lys Phe Phe Pro Asp Ile Ile Lys Ile His Trp Gln Glu Lys 35 40 45Lys Ser Asn Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met Lys Thr 50 55 60Asn Asp Thr Tyr Met Lys Phe Ser Trp Leu Thr Val Pro Glu Glu Ser65 70 75 80Leu Asp Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn Lys Asn 85 90 95Gly Ile Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr 100 105120204PRTArtificial SequenceTRGC2 (3X) 120Lys Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu Pro1 5 10 15Ser Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu 20 25 30Leu Glu Lys Phe Phe Pro Asp Ile Ile Lys Ile His Trp Gln Glu Lys 35 40 45Lys Ser Asn Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met Lys Thr 50 55 60Asn Asp Thr Tyr Met Lys Phe Ser Trp Leu Thr Val Pro Glu Glu Ser65 70 75 80Leu Asp Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn Lys Asn 85 90 95Gly Ile Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val Thr 100 105 110Thr

Val Asp Pro Lys Asp Ser Tyr Ser Lys Asp Ala Asn Asp Val Thr 115 120 125Thr Val Asp Pro Lys Tyr Asn Tyr Ser Lys Asp Ala Asn Asp Val Ile 130 135 140Thr Met Asp Pro Lys Asp Asn Trp Ser Lys Asp Ala Asn Asp Thr Leu145 150 155 160Leu Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr Leu Leu Leu 165 170 175Leu Leu Lys Ser Val Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu Leu 180 185 190Gly Arg Thr Ala Phe Cys Cys Asn Gly Glu Lys Ser 195 200121214PRTArtificial SequenceIGHEP1 121Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp1 5 10 15Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu 20 25 30Ala Pro Ser Lys Trp Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly 35 40 45Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly 50 55 60Thr Leu Thr Val Thr Ser Thr Val Pro Val Gly Thr Arg Asp Trp Ile65 70 75 80Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro Gln Leu Pro Arg 85 90 95Ala Leu Val Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro 100 105 110Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Leu Gly Ser Arg Asp Lys 115 120 125Arg Thr Leu Thr Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser 130 135 140Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser145 150 155 160Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Ile Phe Ser 165 170 175Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile 180 185 190Cys Arg Ala Val His Glu Ala Ala Ile Pro Ser Gln Thr Val Gln Arg 195 200 205Ala Val Ser Val Asn Pro 210122107PRTHomo sapiensMISC_FEATURE(1)..(107)Human IgG1 CH3 domain 122Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105123107PRTHomo sapiensMISC_FEATURE(1)..(107)CH3-BT alpha domain 123Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Lys Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Tyr Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Ser Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105124107PRTHomo sapiensMISC_FEATURE(1)..(107)CH3-BT beta domain 124Gly Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly Ser Phe 50 55 60Cys Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105

Claims

1. A hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof comprising: (a) a first engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said first engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a first member of the naturally occurring immunoglobulin super-family; and (b) a second engineered immunoglobulin chain comprising an engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a second member of the naturally occurring immunoglobulin super-family, wherein the engineered domain of the first engineered immunoglobulin chain and the engineered domain of the second engineered immunoglobulin chain are not identical and, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a CH3 domain, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residue at position 88, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90 and, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residue at position 85.1 and/or 86 and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 86, 88, and 90, wherein the amino acid residue substituted at position 88 in the parent domain of the first engineered immunoglobulin chain is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the parent domain of the second engineered immunoglobulin chain, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

2. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the parent domain of the first and/or the second engineered immunoglobulin chain is a naturally occurring CH3 domain.

3. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid sequence of the engineered domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain do not contain an insertion of one or more amino acid residues compared to the amino acid sequence of the parent domains of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain.

4. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the donor domain of the first member of the naturally occurring immunoglobulin super-family and the donor domain of the second member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer.

5. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is not a charged amino acid or is not 88I.

6. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is 88W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

7. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted at position 85 and/or 86 in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 85.1A, 85.1S, 85.1C and 85.1N and conservative amino acid substitutions thereof and/or is selected from the group consisting of 86S and 86V and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

8. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88Y, 88K and 88 W and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20E, 20S, 20K, 20W, 22A, 22G, 22T, 22L, 22I, 22V, 26R, 26Q, 26T, 26K, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1F, 85.1C, 85.1N, 85.1W, 86S, 86I, 86T, 86H, 86Q, 86V, 86W, 86Y and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

9. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain at position 88 is selected from the group consisting of 88Q, 88L, 88V, 88R, 88E, 88I, 88T, 88Y, and 88W and/or wherein the further amino acid residue substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20E, 20S, 20K, 22A, 22G, 22T, 22L, 22I, 22V, 26Q, 26T, 26K, 26V, 26S, 26N, 26E, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K, 85.1F, 85.1C, 85.1N, 86S, 86I, 86T, 86H, 86Q, 86V, and 86F, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

10. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3E, 3K, 5A, 5T, 7F, 7M, 20K, 20N, 20T, 22A, 22L, 22V, 26E, 26T, 27E, 27K, 79F, 79Y, 81A, 81G, 81D, 84L, 84M, 84.2E, 84.2S, 85.1A, 85.1C, 85.1M, 85.1N, 85.1S, 86F, 86S, 86V, 90K, 90N, and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

11. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain comprises the amino acid residues at position 88 and at position 20, and optionally a further amino acid residue at a position selected from the group consisting of 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

12. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and/or 86 and at position 26 and optionally an amino acid residue at a further position, wherein the amino acid position is indicated according to the IMGT.RTM. numbering.

13. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 20, 22, 26, 79, 85.1, 86, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

14. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 20K, 22V, 26T, 79Y, 85.1S, 86V, and 90N and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

15. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 84 or is 84L and conservative amino acid substitutions thereof, and optionally a further amino acid at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

16. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 81, 84, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

17. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and 86 or comprises 85.1C and conservative amino acid substitutions thereof and 86S and conservative amino acid substitutions thereof, and wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 3E, 5A, 7F, 20T, 22V, 26T, 81D, 84L, 84.2E, 88R and 90R and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

18. (canceled)

19. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is at a position selected from the group consisting of 3, 5, 20, 22, 26, 27, 81, 84, 85.1, and 86, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

20. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is selected from the group consisting of 3K, 5T, 20T, 22L, 26E, 27K, 81G, 84M, 85.1M, 86F and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

21. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 79, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

22. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is at position 79 or is 79F and conservative amino acid substitutions thereof, and optionally a further amino acid residue at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 85.1, 88, and 90 or at a position selected from the group consisting of 7, 20, 22, 27, 81, 84.2, 86, 88, and 90, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

23. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain comprises the amino acid residues at position 85.1 and 86 or comprises 85.1N and conservative amino acid substitutions thereof and 86V and conservative amino acid substitutions thereof, and wherein the further amino acid residue which is substituted in the protein-protein interface of the parent domain of the second engineered immunoglobulin chain is selected from the group consisting of 7M, 20N, 22A, 27E, 79F, 81A, 84.2S, 85.1N, 86V, 88L, and 90K and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

24. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 88I and conservative amino acid substitutions thereof and the optional further amino acid residue which is substituted in the protein-protein interface of the parent domain of the first engineered immunoglobulin chain is 81W and conservative amino acid substitutions thereof, wherein the amino acid position of each group member is indicated according to the IMGT.RTM. numbering.

25-55. (canceled)

56. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the amino acid residues of the protein-protein interface of the parent domain of the first and/or second engineered immunoglobulin chain which are substituted are not adjacent.

57. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein at least two amino acid residues of the protein-protein interface of the parent domain of the first and/or second engineered immunoglobulin chain are substituted.

58-61. (canceled)

62. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain comprise a further engineered domain with a protein-protein interface, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said further engineered domain of said first engineered immunoglobulin chain and/or said second engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a third member of the naturally occurring immunoglobulin super-family, and wherein the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain is interacting with the protein-protein interface of an engineered domain of a third engineered immunoglobulin chain by hetero-dimerization or by homo-dimerization, wherein at least one amino acid residue of the protein-protein interface of the parent domain of said engineered domain of said third engineered immunoglobulin chain is substituted with an amino acid residue at the equivalent 3D structural position from a protein-protein interface of a donor domain of a fourth member of the naturally occurring immunoglobulin super-family, and wherein the donor domain of the third member of the naturally occurring immunoglobulin super-family and the donor domain of the fourth member of the naturally occurring immunoglobulin super-family form a naturally occurring hetero-dimer or a naturally occurring homo-dimer and, wherein the protein-protein interface of the further engineered domain of the first engineered immunoglobulin chain and/or the second engineered immunoglobulin chain and of the engineered domain of the third engineered immunoglobulin chain is different from the protein-protein interface of the engineered domain of the first engineered immunoglobulin chain and of the engineered domain of the second engineered immunoglobulin chain.

63-74. (canceled)

75. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the first and second engineered immunoglobulin chains comprise an Fc region.

76. (canceled)

77. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein the hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof is a full-length antibody.

78-79. (canceled)

80. The hetero-dimeric immunoglobulin or hetero-dimeric fragment thereof of claim 1, wherein at least one additional polypeptide is fused to the first and/or second engineered immunoglobulin chain.

81-211. (canceled)