HUMANIZED, ANTI-N2 ANTIBODIES

Abstract

The present invention encompasses humanized antibodies that specifically bind N2 peptide, methods for the preparation thereof and methods for the use thereof.

Description

RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. application Ser. No. 14/206,368, filed on Mar. 12, 2014, which claims the benefit of U.S. Provisional Application No. 61/777,647, filed Mar. 12, 2013. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] It has been demonstrated that ischemia-reperfusion injury can be initiated by clonally-specific pathogenic IgM that activates the classical pathway of complement (Zhang et al. (2004) Proc. Natl. Acad. Sci. 101(11):3886-3891). Pathogenic IgM (also referred to as "natural IgM") recognizes and binds to a self-antigen which is an antigen expressed or exposed on damaged tissue, for example, on damaged ischemic tissue. Binding of pathogenic IgM to the self-antigen initiates inflammation by activating complement in the classical pathway. U.S. Pat. No. 7,442,783 describes the major epitope for binding of natural IgMs as a conserved region within type II non-muscle myosin heavy chain (NMHC) proteins. This epitope is referred to as the N2 12-mer peptide.

[0004] Inhibitors of the interaction between the N2 epitope and pathogenic IgM have been described as useful for the treatment of inflammatory diseases and conditions, including ischemia/reperfusion injury. For example, U.S. Pat. No. 8,324,352 describes the murine monoclonal antibody referred to as 21G6. Murine 21G6 (m21G6) was shown to bind to the N2 peptide and provide protection against ischemia/reperfusion injury in animal models. It would be advantageous to develop additional therapeutic agents that bind the N2 peptide and that can be used for treating inflammatory conditions such as ischemia/reperfusion injury.

SUMMARY OF THE INVENTION

[0005] The present invention encompasses humanized derivatives of the murine 21G6 antibody that specifically bind N2 peptide. As shown in the Examples below, humanized antibodies have been developed that bind the N2 peptide.

[0006] In one embodiment, the invention is directed to an antibody or antigen binding fragment thereof comprising framework regions from a human immunoglobulin and comprising the variable heavy chain (VH) complementarity determining regions (CDRs) of the murine 21G6 antibody and the variable light chain (VL) CDRs of the murine 21G6 antibody.

[0007] In some embodiments, the invention is directed to a humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0008] i. the VH region comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 comprises SEQ ID NO: 3, VH CDR2 comprises SEQ ID NO: 4 and VH CDR3 comprises SEQ ID NO: 5;

[0009] ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:

[0010] a. the VH FWR1 comprises SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23;

[0011] b. The VH FWR2 comprises SEQ ID NO: 16, SEQ ID NO: 20 or SEQ ID NO: 24;

[0012] c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45; and

[0013] d. VH FWR4 comprises SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26;

[0014] iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3, wherein the VL CDR1 comprises SEQ ID NO: 6, VL CDR2 comprises SEQ ID NO: 7 and VL CDR3 comprises SEQ ID NO: 8;

[0015] iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:

[0016] a. the VL FWR1 comprises SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35;

[0017] b. VL FWR2 comprises SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36;

[0018] c. VL FWR3 comprises SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and

[0019] d. VL FWR4 comprises SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

[0020] In certain additional embodiments, the antibody or antigen-binding fragment has a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In yet additional embodiments, the antibody or antigen-binding fragment has a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49.

[0021] In additional aspects, the antibody or antigen-binding fragment has a VL region that comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0022] In yet additional aspects, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 9 and has a VL region that consists of SEQ ID NO: 12. In other embodiments, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 9 and has a VL region that consists of SEQ ID NO: 13. In another aspect, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 9 and the VL region consists of SEQ ID NO: 14. In a further embodiment, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 10 and has a VL region consists of SEQ ID NO: 12. In certain additional aspects, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 10 and has a VL region that consists of SEQ ID NO: 13. In an additional embodiment, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 10 and has a VL region that consists of SEQ ID NO: 14. In another aspect of the invention, the antibody or antigen-binding fragment of has a VH region that consists of SEQ ID NO: 11 and a VL region that consists of SEQ ID NO: 12. In another embodiment, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 11 and a VL region that consists of SEQ ID NO: 13. In another embodiment, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 11 and a VL region that consists of SEQ ID NO: 14. In yet additional embodiments, the antibody or antigen-binding fragment of has a VH region that consists of SEQ ID NO: 49 and a VL region that consists of SEQ ID NO: 12. In another embodiment, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 49 and a VL region that consists of SEQ ID NO: 13. In another embodiment, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 49 and a VL region that consists of SEQ ID NO: 14.

[0023] In some embodiments, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 43 and a VL region that consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In additional embodiments, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 44 and a VL region that consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In yet other embodiments, the antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 45 and a VL region that consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In another aspect, the invention is an antibody or antigen-binding fragment has a VH region that consists of SEQ ID NO: 42 and VL region consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0024] In yet additional embodiments, the invention is directed to a humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region comprising a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In an additional embodiment, the invention is directed to a humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region comprising a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49.

[0025] In a further embodiment, the invention is a humanized, anti-N2 antibody or antigen-binding fragment thereof, comprising a light chain variable (VL) region comprising a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0026] In certain embodiments, the invention is directed to a humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0027] i. the VH region comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 comprises SEQ ID NO: 3, VH CDR2 comprises SEQ ID NO: 4 and VH CDR3 comprises SEQ ID NO: 5;

[0028] ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:

[0029] a. the VH FWR1 comprises SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23;

[0030] b. The VH FWR2 comprises SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:24 or SEQ ID NO: 50;

[0031] c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and

[0032] d. VH FWR4 comprises SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26;

[0033] iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3 wherein the VL CDR1 comprises SEQ ID NO: 6, VH CDR2 comprises SEQ ID NO: 7 and VH CDR3 comprises SEQ ID NO: 8;

[0034] iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:

[0035] a. the VL FWR1 comprises SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35;

[0036] b. VL FWR2 comprises SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36;

[0037] c. VL FWR3 comprises SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and

[0038] d. VL FWR4 comprises SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

[0039] In certain additional embodiments, the antibody or antigen-binding fragment has a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49. In additional aspects, the antibody or antigen-binding fragment has a VL region that comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0040] In yet additional embodiments, the antibody or antigen binding fragment has a VH that consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 12. In yet other embodiments, the antibody or antigen-binding fragment has a VH region that consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 13. In yet further embodiments, the antibody or antigen-binding fragment has a VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 14. In yet an additional aspect, the antibody or antigen-binding fragment has a VH region that consists of the amino acid sequence of SEQ ID NO: 54 and VL region consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0041] In further embodiments, the invention is directed to a nucleotide encoding the humanized, anti-N2 antibody or antigen-binding fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale emphasis instead being placed upon illustrating the principles of the invention.

[0043] FIG. 1 shows a sequence comparison of the murine 21G6 heavy chain variable 5 (VH) region (SEQ ID NO: 1) and the humanized heavy chain variable regions (VH) H1, H2 and H3 (SEQ ID NOS: 9-11, respectively) and also shows a sequence comparison of the murine 21G6 light chain variable (VL) region (SEQ ID NO: 2) and the humanized light chain variable regions light chain (VL) regions L1, L2 and L3 (SEQ ID NOS: 12-14, respectively). The CDR regions are indicated by the boxes.

[0044] FIG. 2 shows a sequence comparison of murine 21G6 heavy chain (VH) region (SEQ ID NO: 1) and the humanized heavy chain variable region H4 (SEQ ID NO: 49).

[0045] FIG. 3 shows a nucleotide sequence (SEQ ID NO: 60) encoding the humanized heavy chain variable region H4 that was optimized for the production of the humanized 20 antibody in Chinese hamster ovary (CHO) cells. FIG. 3 also shows the amino acid sequence of the heavy chain variable region H4 (SEQ ID NO: 54). Each + indicates where a change was made as compared to (SEQ ID NO: 66).

[0046] FIG. 4 shows a nucleotide sequence (SEQ ID NO: 69) encoding the humanized light chain variable region L2 that was optimized for the production of the humanized antibody in Chinese hamster ovary (CHO) cells. FIG. 4 also shows the amino acid of the light chain variable region L2 (SEQ ID NO: 55). Each + indicates where a change was 30 made as compared to (SEQ ID NO: 67).

[0047] FIG. 5 is a bar graph showing the levels of recombinant antibody expression on day 5 post-transfection with either the native sequences (pDS2.0; bars on the right) or optimized sequences (pDS-opt; bars on the left). Incorporation of the recombinant antibody into the CHO cells was accomplished by selection in the presence of 10 ug/ml puromycin and 100 nM methotrexate for transfectants DS5 and DS7 or 20 ug/ml puromycin/200 nM methotrexate for transfectants DS6 and DS8.

DETAILED DESCRIPTION OF THE INVENTION

[0048] A description of preferred embodiments of the invention follows.

[0049] The words "a" or "an" are meant to encompass one or more, unless otherwise specified.

[0050] An "antibody" is a binding molecule including immunoglobulin molecules, antibody fragments, and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen-binding site. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains. Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end. An antibody binds specifically to an antigen (or other molecule) if the antibody binds preferentially to the antigen, and, for example, has less than about 30%, preferably less than about 20%, less than about 10%, or less than about 1% cross-reactivity with another molecule. The terms "antibody" and "immunoglobulin" are used interchangeably. "Bind" or "binding" are used herein to refer to detectable relationships or associations (e.g. biochemical interactions) between molecules.

[0051] An "isolated" molecule, for example, an isolated antibody or isolated peptide, refers to a condition of being separate or purified from other molecules present in the natural environment or as they occur in nature.

[0052] The N2 epitope is an epitope of the self-antigen, the 12 amino acid sequence expressed in non-muscle myosin heavy chain (NMHC) type II. The 12-amino acid sequence is LMKNMDPLNDNV (SEQ ID NO: 47). The N2 epitope is described in detail in U.S. Pat. No. 7,442,783, the contents of which are expressly incorporated by reference herein. "Natural IgM" or "pathogenic IgM" refers to an IgM antibody that is naturally produced in a mammal (for example, a human) that binds to the N2 epitope and initiates inflammation by activating complement in the classical pathway.

[0053] In some embodiments, antibody or antigen-binding fragment thereof binds to SEQ ID NO: 47. In additional embodiments, the antibody or antigen-binding fragment thereof binds to an epitope wherein the amino acid sequence of the epitope has at least about 80%, 85%, 90%, 95%, or 98% sequence identity to the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the epitope comprises the amino acid sequence LMKNMDPLNDNI (SEQ ID NO: 48).

[0054] The hypervariable region of an antibody or fragment thereof refers to the amino acid residues that contribute to antigen-binding. The hypervariable region comprises amino acid residues from the complementarity determining regions (CDRs). The CDRs are specific regions within variable regions of the heavy and the light chain. Generally, the variable region consists of four framework regions (FWR1, FWR2, FWR3, FWR4) and three CDRs arranged as follows: NH.sub.2-FWR1-CDR1-FWR2-CDR2-FWR3-CDR3-FWR4-constant region-C(O)OH. The term "framework regions" refers to those variable domain amino acid residues other than the CDR residues and include, for example, FWR1, FWR2, FWR3, and FWR4.

[0055] As described above, the present invention is directed to humanized derivatives of the murine 21G6 antibody described in U.S. Pat. No. 8,324,352, the contents of which are expressly incorporated herein. In certain embodiments, the humanized antibodies and fragments thereof bind the N2 peptide. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the murine 21G6 antibody are shown in FIG. 1 and are below as SEQ ID NOs: 1 and 2:

TABLE-US-00001 Murine 21G6 (m21G6) VH (SEQ ID NO: 1) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGG INPSNGGTNFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRWG YDREWFAYWGQGTLVTVSA. Murine 21G6 VL (SEQ ID NO: 2) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQ VLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYP FTFGSGTKLEIKR.

The underlined amino acids represent the complementarity determining regions.

[0056] FIG. 1 shows the amino acid sequences of three VH regions encompassed by the invention: H1-21G6, H2-21G6 and H3-21G6. The amino acid sequences of the H1-21G6, H2-21G6 and H3-21G6 VH regions are SEQ ID NOs: 9, 10 and 11, respectively:

TABLE-US-00002 H1-21G6 VH (SEQ ID NO: 9) QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYYMYWVKQAPGQGLEWIGG INPSNGGTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRWG YDREWFAYWGQGTLVTVSS. H2-21G6 VH (SEQ ID NO: 10) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKSKATMTVDKSTSTAYMELRSLRSDDSAVYYCTRWG YDREWFAYWGQGTLVTVSS. H3-21G6 VH (SEQ ID NO: 11) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKSKATITVDKSTSTAYMELSSLRSEDTAVYYCTRWG YDREWFAYWGQGTLVTVSS.

The underlined amino acids represent the complementarity determining regions.

[0057] An additional VH region is also encompassed by the invention: H4-21G6. The amino acid sequence of H4-21G6 is shown below:

TABLE-US-00003 H4-21G6 Vh (SEQ ID NO: 49) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWRQAPGQGLEWMGGI NPSNGGTNFNEKFKSRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRWGY DREWFAYAVGQGTLVTVSS

[0058] The Figure also shows the amino acid sequences of three VL regions encompassed by the invention: L1-21G6, L2-21G6 and L3-21G6. The amino acid sequences of L1-21G6, L2-21G6 and L3-21G6 VH regions are SEQ ID NOs: 12, 13 and 14, respectively.

TABLE-US-00004 L1-21G6 VL (SEQ ID NO: 12) DIVMTQSPATLSVSPGERATISCRSSKSLLHSNGNTYLYWFQQKPGQPPK VLIYRMSNLASGVPARFSGSGSGTDFTLTISSVEPEDFATYYCMQHLEYP FTFGGGTKLEIKR. L2-21G6 VL (SEQ ID NO: 13) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYP FTFGQGTKLEIKR. L3-21G6 VL (SEQ ID NO: 14) DIVMTQTPLSLSYTPGQPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYP FTFGQGTKLEIKR.

The underlined amino acids represent the complementarity determining regions.

[0059] The names "H1-21G6," "H2-21G6," "H3-21G6" and "H4-21G6" are used interchangeably herein with "H1," "H2," "H3," and "H4," respectively. The names "L1-21G6," "L2-21G6" and "L3-21G6" are used interchangeably with "L1," "L2" and "L3," respectively.

[0060] CDR1, CDR2 and CDR3 of the VH regions of the antibodies or fragments of the present invention are SYYMY (SEQ ID NO: 3), GINPSNGGTNFNEKFKS (SEQ ID NO: 4), GYDREWFAY (SEQ ID NO: 5), respectively. CDR1, CDR2 and CDR3 of the VL regions of the antibodies or fragments of the present invention are RSSKSLLHSNGNTYLY (SEQ ID NO: 6), RMSNLAS (SEQ ID NO: 7), and MQHLEYPFT (SEQ ID NO: 8), respectively. The VL region of the antibody or antigen-binding fragments of the present invention includes at least two of the CDRs of m21G6 VL. The VH region of the antibody or antigen-binding fragment of the invention includes at least two CDRs of the m21G6 VH. In some embodiments, the humanized antibodies include all three CDRs of m21G6 VH and/or all three CDRs of the m21G6 VL. The framework regions FWR1, FWR2, FWR3 and FWR4 of the VH region of each of H1-21G6, H2-21G6 and H3-21G6 are shown below:

TABLE-US-00005 H1 VH FWR1 (SEQ ID NO: 15) QVQLVQSGAEVVKPGASVKLSCKASGYTFT. H1 VH FWR2 (SEQ ID NO: 16) WVKQAPGQGLEWIG. H1 VH FWR3 (SEQ ID NO: 17) KATLTVDKSASTAYMELSSLRSEDTAVYYCTR. H1 VH FWR4 (SEQ ID NO: 18) WGQGTLVTVSS. H2 VH FWR1 (SEQ ID NO: 19) QVQLVQSGAEVKKPGASVKVSCKASGYTFT. H2 VH FWR2 (SEQ ID NO: 20) WVRQAPGQGLEWIG. H2 VH FWR3 (SEQ ID NO: 21) KATMTVDKSTSTAYMELRSLRSDDSAVYYCTR. H2 VH FWR4 (SEQ ID NO: 22) WGQGTLVTVSS. H3 VH FWR1 (SEQ ID NO: 23) QVQLVQSGAEVKKPGSSVKVSCKASGYTFT. H3 VH FWR2 (SEQ ID NO: 24) WVRQAPGQGLEWIG. H3 VH FWR3 (SEQ ID NO: 25) KATITVDKSTSTAYMELSSLRSEDTAVYYCTR. H3 VH FWR4 (SEQ ID NO: 26) WGQGTLVTVSS. H4 VH FWR1 (SEQ ID NO: 19) QVQLVQSGAEVKKPGASVKVSCKASGYTFT. H4 VH FWR2 (SEQ ID NO: 50) WVRQAPGQGLEWMG. H4 VH FWR3 (SEQ ID NO: 51) RVTMTTDTSTSTAYMELRSLRSDDTAVYYCTR. H4 VH FWR4 (SEQ ID NO: 22) WGQGTLVTVSS.

The framework regions FWR1, FWR2, FWR3 and FWR4 of each of the VL region of each of L1-21G6, L2-21G6 and L3-21G6 are shown below:

TABLE-US-00006 L1 VL FWR1 (SEQ ID NO: 27) DIVMTQSPATLSVSPGERATISC. L1 VL FWR2 (SEQ ID NO: 28) WFQQKPGQPPKVLIY. L1 VL FWR3 (SEQ ID NO: 29) GVPARFSGSGSGTDFTLTISSVEPEDFATYYC. L1 VL FWR4 (SEQ ID NO: 30) FGGGTKLEIKR. L2 VL FWR1 (SEQ ID NO: 31) DIVMTQSPLSLPVTPGEPASISC. L2 VL FWR2 (SEQ ID NO: 32) WFLQKPGQSPQLLIY. L2 VL FWR3 (SEQ ID NO: 33) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC. L2 VL FWR4 (SEQ ID NO: 34) FGQGTKLEIKR. L3 VL FWR1 (SEQ ID NO: 35) DIVMTQTPLSLSYTPGQPASISC. L3 VL FWR2 (SEQ ID NO: 36) WFLQKPGQSPQLLIY. L3 VL FWR3 (SEQ ID NO: 37) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC. L3 VL FWR4 (SEQ ID NO: 38) FGQGTKLEIKR.

[0061] As described above, the present invention encompasses an antibody or antigen-binding fragment thereof comprising VH CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and VL CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and further comprising a VH region that comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:

[0062] a. the VH FWR1 comprises SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23;

[0063] b. The VH FWR2 comprises SEQ ID NO: 16, SEQ ID NO: 20 or SEQ ID NO:24;

[0064] c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45; and

[0065] d. VH FWR4 comprises SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; and a VL region that comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:

[0066] a. the VL FWR1 comprises SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35;

[0067] b. VL FWR2 comprises SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36;

[0068] c. VL FWR3 comprises SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and

[0069] d. VL FWR4 comprises SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

[0070] The present invention additionally encompasses an antibody or antigen-binding fragment thereof comprising VH CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and VL CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and further comprising a VH region that comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:

[0071] a. the VH FWR1 comprises SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23;

[0072] b. The VH FWR2 comprises SEQ ID NO: 16, SEQ ID NO: 20 or SEQ ID NO:24 or SEQ ID NO: 50;

[0073] c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and

[0074] d. VH FWR4 comprises SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; and a VL region that comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:

[0075] a. the VL FWR1 comprises SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35;

[0076] b. VL FWR2 comprises SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36;

[0077] c. VL FWR3 comprises SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and

[0078] d. VL FWR4 comprises SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

[0079] The terms "comprises" and "comprising" permits (but does not require) the inclusion of additional elements. For example, in the context of an amino acid sequence, the terms "comprises" and "comprising" permits the inclusion of additional amino acids at either the N-terminus and/or the carboxy terminal end. In some embodiments, the framework region of the VH and VL regions comprise a specific indicated amino acid sequence and one to three additional amino acids at the N-terminus and/or at the carboxy terminal end.

[0080] In certain additional aspects, the antibody or antigen binding fragment of the invention comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0081] i. the VH comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 consists of SEQ ID NO:3, VH CDR2 consists of SEQ ID NO: 4 and VH CDR3 consists of SEQ ID NO: 5;

[0082] ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:

[0083] a. the VH FWR1 consists of SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23;

[0084] b. The VH FWR2 consists of SEQ ID NO: 16, SEQ ID NO: 20 or SEQ ID NO:24;

[0085] c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45; and

[0086] d. VH FWR4 consists of SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26;

[0087] iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3 wherein the VL CDR1 consists of SEQ ID NO: 6, VH CDR2 comprises SEQ ID NO: 7 and VH CDR3 consists of SEQ ID NO: 8;

[0088] iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:

[0089] a. the VL FWR1 consists of SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35;

[0090] b. VL FWR2 consists of SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36;

[0091] c. VL FWR3 consists of SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37;

[0092] d. VL FWR4 consists of SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

[0093] In yet additional embodiments, the present invention additionally encompasses an antibody or antigen-binding fragment thereof comprising VH CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and VL CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and further comprising a VH region that comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:

[0094] a. the VH FWR1 consists of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23;

[0095] b. The VH FWR2 consists of SEQ ID NO: 16, SEQ ID NO: 20 or SEQ ID NO:24 or SEQ ID NO: 50;

[0096] c. VH FWR3 consists of SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and

[0097] d. VH FWR4 consists of SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; and a VL region that comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:

[0098] e. the VL FWR1 consists of SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35;

[0099] f. VL FWR2 consists of SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36;

[0100] g. VL FWR3 consists of SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and

[0101] h. VL FWR4 consists of SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

[0102] As described above, FIG. 1 shows the amino acid sequences of three exemplary humanized VH regions that comprise the VH CDRs of m21G6 (H1-21G6, H2-21G6 and H3-21G6; SEQ ID NOs: 9, 10 and 11, respectively). An additional exemplary humanized VH region that comprises the VH CDRs of m21G6 is H4-21G6 (SEQ ID NO: 49 or SEQ ID NO: 54). FIG. 1 also shows the amino acid sequences of three exemplary humanized VL regions (L1-21G6, L2-21G6 and L3-21G6; SEQ ID NOs: 12, 13 and 14, respectively). In some embodiments, the antibody or antigen-binding fragments of the invention comprise a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In yet an additional embodiment, the antibody or antigen-binding fragment comprises a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 49 and SEQ ID NO: 54, and comprises a VL region that comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In additional embodiments, the antibody or antigen-binding fragments of the invention comprise a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 49 and SEQ ID NO: 54. In additional aspects, the antibody or antigen-binding fragment has a VL region that comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. In yet another embodiment, the antibody or antigen-binding fragment comprises a VH region that comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 and comprises a VL region that comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0103] In some embodiments, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 9 and has a VL region that comprises or consists of SEQ ID NO: 12. In other embodiments, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 9 and has a VL region that comprises or consists of SEQ ID NO: 13. In another aspect, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 9 and the VL region comprises or consists of SEQ ID NO: 14. In a further embodiment, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 10 and has a VL region that comprises or consists of SEQ ID NO: 12. In certain additional aspects, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 10 and has a VL region that comprises or consists of SEQ ID NO: 13. In an additional embodiment, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 10 and has a VL region that comprises or consists of SEQ ID NO: 14. In another aspect of the invention, the antibody or antigen-binding fragment of claim 1 has a VH region that comprises or consists of SEQ ID NO: 11 and a VL region that comprises or consists of SEQ ID NO: 12. In another embodiment, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 11 and a VL region that comprises or consists of SEQ ID NO: 13. In another embodiment, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 11 and a VL region that comprises or consists of SEQ ID NO: 14.

[0104] In yet additional embodiments, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 49 and has a VL region that comprises or consists of SEQ ID NO: 12. In another aspect, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 49 and the VL region comprises or consists of SEQ ID NO: 13. In a further embodiment, the antibody or antigen-binding fragment has a VH region that comprises or consists of SEQ ID NO: 49 and has a VL region that comprises or consists of SEQ ID NO: 14.

[0105] In certain additional embodiments, the antibody or antigen-binding fragment comprises a VH region that comprises a sequence selected from SEQ ID NO: 49 and SEQ ID NO: 54. In yet additional embodiments, the antibody or antigen-binding fragment comprises a VL region that comprises SEQ ID NO: 13 or SEQ ID NO: 55. In a further embodiment, the antibody or antigen-binding fragment comprises a VH region that comprises SEQ ID NO: 49 or SEQ ID NO: 54 and a VL region that comprises SEQ ID NO: 13 or SEQ ID NO: 55. In an additional aspect, the antibody or antigen-binding fragment comprising a VH region that comprises SEQ ID NO: 49 and a VL region that comprises SEQ ID NO: 13.

[0106] In certain aspects of the invention, the isotype of the constant region of the antibodies or antigen-binding fragments of the invention is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the isotype of the IgG constant region is IgG1. In other embodiments, the isotype of the IgG constant region is IgG4. In some embodiments, the antibody or antigen-binding fragment thereof have a human IgG1 constant domain or a human IgG4 constant domain. In additional aspects, the antibody or antigen-binding fragment has a human Ig kappa constant domain. The term "isotype" refers to the classification of an antibody's heavy or light chain constant region. The constant domains of antibodies are not involved in binding to antigen, but have various effector functions. Depending on the amino acid sequence of the heavy chain constant region, a human or humanized antibody can be deemed to belong to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Many of these classes of immunoglobulins, for example the IgG class, can be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, and IgG4. Human light chain constant regions are classified into two major classes, kappa and lambda.

[0107] When the positions of amino acid residues are referred to by number herein, it is to be understood that Kabat numbering system is used, unless otherwise indicated. Kabat numbering is described in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Publication No. 91-3242, National Institutes of Health, National Technical Information Service (hereinafter "Kabat"). Immunoglobulin sequences can be numbered according to Kabat by performing an alignment with the Kabat reference sequence. As such, the Kabat numbering system provides a uniform system for numbering immunoglobulin chains.

[0108] The present invention is directed to humanized antibodies wherein the CDRs are from the murine 21G6 antibody and wherein the framework regions are from a human immunoglobulin. It will be understood, that humanized antibodies can comprise amino acid residues that are not found in the recipient antibody or in the donor antibody. For example, such changes in the amino acid sequence can be made to improve binding to the antigen (for example, the N2 peptide) and/or to reduce immunogenicity. Therefore, the present invention encompasses the antibodies or antigen-binding fragments described herein wherein specific amino acids have been substituted, deleted or added. Amino acid substitutions, deletions or additions can be made to the antibodies or antigen-binding fragments thereof to improve or refine the properties of the antibody or fragment, for example amino acid change can be made to inhibit or block inflammation. For example, asparagine at position 297 (Asn 297) of the IgG constant region can be replaced with an alternative amino acid to reduce glycosylation and decrease activation of complement and binding to the Fc receptor. See, for example, Leatherbarrow et al. (1985) Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor. Mol Immunol 22(4):407-415; Tao et al. (1989) Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol 143(8):2595-2601; Kabat (1987) Sequences of Proteins of Immunological Interest (In: US Department of Human Services), and Sazinsky et al. (2008), Aglycosylated immunoglobulin G.sub.1 variants productively engage activating Fc receptors, PNAS 105(51): 20167-20172, the contents of each of which are expressly incorporated by reference herein. Glycosylation can be reduced, for example, by replacing the asparagine at position 297 (Asn 297) with an alternative amino acid, for example, alanine, glutamine, histidine or glycine. In some embodiments, Asn 297 can be replaced with glutamine. In certain aspects, the antibody or antigen-binding fragment has a human IgG1 constant domain that is aglycosylated.

[0109] In some embodiments, the penultimate amino acid in the third framework of the VH (VH FWR3) of each of H1-21G6, H2-21G6 and H3-21G6 (SEQ ID NOs: 17, 21 and 25, respectively) can be changed from threonine to alanine. The amino acid sequences SEQ ID NOs: 39, 40 and 41 are sequences for the VH FWR3 of each of H1, H2 and H3 wherein the penultimate amino acid (threonine) has been replaced with alanine:

TABLE-US-00007 H1 VH FWR3 with amino acid mutation to alanine (SEQ ID NO: 39) KATLTVDKSASTAYMELSSLRSEDTAVYYCAR. H2 VH FWR3 with amino acid mutation to alanine (SEQ ID NO: 40) KATMTVDKSTSTAYMELRSLRSDDSAVYYCAR. H3 VH FWR3 with amino acid mutation to alanine (SEQ ID NO: 41) KATITVDKSTSTAYMELSSLRSEDTAVYYCAR.

The italicized alanine above represents the change from threonine to alanine. The amino acid sequences SEQ ID NOs: 43, 44, and 45 are sequences for the H1, H2 and H3 VH regions wherein the penultimate amino acid (threonine) of the FWR3 is replaced with alanine:

TABLE-US-00008 HI VH with amino acid mutation to alanine in FWR3 (SEQ ID NO: 43) QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYYMYWVKQAPGQGLEWIGG INPSNGGTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCARWG YDREWFAYWGQGTLVTVSS. H2 VH with amino acid mutation to alanine in FWR3 (SEQ ID NO: 44) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIG GINPSNGGTNFNEKFKSKATMTVDKSTSTAYMELRSLRSDDSAVYYCAR WGYDREWFAYWGQGTLVTVSS. H3 with amino acid mutation to alanine in FWR3 (SEQ ID NO: 45) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIG GINPSNGGTNFNEKFKSKATITVDKSTSTAYMELSSLRSEDTAVYYCAR WGYDREWFAYWGQGTLVTVSS.

The italicized alanine above represents the change from threonine to alanine.

[0110] In an additional embodiment, the penultimate amino acid in the third framework regions of the VH (VH FWR3) of H4-21G6 (SEQ ID NO: 51) can be changed from threonine to alanine. The amino acid sequence SEQ ID NO: 52 is the sequence for the VH FWR3 of H4 wherein the penultimate amino acid (threonine has been replaced with alanine:

TABLE-US-00009 H4 VH FWR3 (SEQ ID NO: 52) RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR.

The italicized alanine above represents the change from threonine to alanine. The amino acid sequence SEQ ID NO: 52 is the sequence for the H4 VH regions wherein the penultimate amino acid (threonine) of the FWR3 is replaced with alanine:

TABLE-US-00010 H4 VH with amino acid mutation to alanine in FWR3 (SEQ ID NO: 53) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMG GINPSNGGTNFNEKFKSRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR WGYDREWFAYWGQGTLVTVSS.

The italicized alanine above represents the change from threonine to alanine.

[0111] Amino acid modifications that may increase stability and/or increase affinity are also contemplated herein. Additional specific amino acid variants contemplated by the invention are variants of H2-21G6 VH and a variant of the murine 21G6 VL kappa chain:

TABLE-US-00011 Amino Acid Variant of H2 VH (SEQ ID NO: 42) QVQLVQSGAELVKKPGASLKVSCKASGYTFTSYYMYWVRQAPGQGLEWIG GINPSNGGTNFNEKFKGRVTITRDKSTSTAYMELRSLRSEDSAVYYCARW GYDREWFAYWGQGTLVTVSS. Amino Acid Variant of kappa chain (m21G6 VL) (SEQ ID NO: 46) EIVLTQSPGTLSLS PGERATLSCRASKSLLHSNGNTYLYWYQQKPGQAP RLLIYRMSNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYC MQHLEYPFTFGQGTKLEIKR.

[0112] Additional amino acid modifications include amino acid variants of the H2 VH amino acid sequence (SEQ ID NO: 42), wherein the amino acid at position 65 is replaced with glycine, the amino acid at position 66 is replaced with arginine, the amino acid at position 67 is replaced with valine or phenylalanine, the amino acid at position 69 is replaced with isoleucine, the amino acid at position 71 is replaced with arginine and/or the amino acid at position 85 can be replaced with glutamic acid.

[0113] The invention also encompasses an antibody or antigen-binding fragment thereof wherein an alanine at position 78 (Ala 78) of the VH is replaced with phenylalanine.

[0114] In certain embodiments, the antibody or antigen-binding fragment has a human IgG4 constant domain wherein serine at position 228 (Ser 228) is replaced with proline.

[0115] Additional modifications can also be made within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, protein stability and/or antigen-dependent cellular cytotoxicity, or lack thereof. In addition, an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody). In addition, the class of an antibody can be "switched" by known techniques. Such techniques include, e.g., the use of direct recombinant techniques (see e.g., U.S. Pat. No. 4,816,397) and cell-cell fusion techniques (see e.g., U.S. Pat. No. 5,916,771). For example, an antibody that was originally produced as an IgM molecule may be class switched to an IgG antibody. Class switching techniques also may be used to convert one IgG subclass to another, e.g., from IgG1 to IgG2. Thus, the effector function of the antibodies of the invention may be changed by isotype switching to, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. Exemplary cDNA sequences for constant regions are available from GenBank, for example, each of which incorporated by reference in its entirety, are as follows: Human IgG1 constant heavy chain region: GenBank Accession No.: J00228; Human IgG2 constant heavy chain region: GenBank Accession No.: J00230; Human IgG3 constant heavy chain region: GenBank Accession No.: X04646; Human IgG4 constant heavy chain region: GenBank Accession No.: K01316; and Human kappa light chain constant region: GenBank Accession No.: J00241. The hinge region of CH.sub.1 can also be modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. The Fc hinge region of an antibody can also be mutated to decrease the biological half-life of the antibody. In another embodiment, the antibody is modified to increase its biological half-life. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effecter function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both to Winter et al. In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al. In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al. In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc.gamma. receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgG1 for Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604).

[0116] In addition, the half-life of an antibody molecule can be increased by increasing the affinity of the antibody for the Fc receptor (FcR). The binding of the antibody to the FcR can be improved using art-known techniques, such as by introducing specific mutations in the Fc region (Dall'Acqua, W. F. et al. (2006) J. Biol. Chem. 281: 23514-23524; Petkova, S. B. (2006) et al., Internat. Immunol. 18:1759-1769; U.S. Pat. Nos. 7,785,791, 7,790,858 and 7,371,826; the contents of each of the aforementioned references are incorporated by reference herein). It has been demonstrated that the increased affinity of the antibody for the FcR results in a longer half-life (Petkova et al., 2006). In the case of amino acid mutagenesis in the Fc domain, it is known that certain amino acid mutations can increase binding to the FcR and extend the half-life. Examples of such amino acid mutations that have been described to increase binding to the Fc receptor include N434A and T307/E380/N434 (Petkova et al., 2006) and as well other mutations M252Y/S254T/T256E, T250Q, M428L, and T250Q/M428L, (Dall'Acqua et al., 2006; Hinton et al., J. Immunol., 176: 346-356).

[0117] An antibody or antigen-binding fragment described herein can be chemically modified based on linkage to a polymer. The polymer is typically water soluble so that the antibody to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer can have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled. An exemplary reactive aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C.sub.1-C.sub.10 alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714). The polymer can be branched or unbranched. For therapeutic use of the end-product preparation, the polymer is pharmaceutically acceptable. The water soluble polymer, or mixture thereof if desired, can be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol.

[0118] An antibody fragment or antigen-binding fragment is a derivative of an antibody that is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, minibody, Fc, Fd fragments, and single chain antibodies.

[0119] Antibody fragments can be produced by methods known in the art. For example, the antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody, or the fragment can be produced recombinantly. The antibody fragment can optionally be a single chain antibody fragment. Alternatively, the fragment can comprise multiple chains which are linked together, for instance, by disulfide linkages. In addition, digestion of an antibody with pepsin yields F(ab').sub.2 fragments and multiple small fragments. Mercaptoethanol reduction of an antibody yields individual heavy and light chains. Digestion of an antibody with papain yields individual Fab fragments and the Fc fragment. The fragment can also optionally be a multimolecular complex. A functional antibody fragment can for example comprise at least about 50 amino acids. In some embodiments, the functional antibody fragment can comprise at least about 200 amino acids.

[0120] Humanized antibodies and antigen-binding fragment thereof described herein can be produced using techniques known in the art, including, but not limited to, CDR-grafting (see, for example, European Patent No. EP 239,400; WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, the contents of each of which incorporated by reference), veneering or resurfacing (see, for example, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci., 91:969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, PCT Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al, Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp): 5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated by reference herein.

[0121] The humanized antibody can be produced by, for example, by constructing cDNAs encoding the humanized variable regions, inserting each of them into an expression vector for animal cells comprising genes encoding the heavy chain and light chain of a human antibody to thereby construct a vector for expression of humanized antibody, and introducing it into an animal cell to express and produce the humanized antibody. The invention encompasses a nucleotide sequence that encodes an antibody or antigen-binding fragment described herein. In additional embodiments, the invention is directed to a nucleotide sequence that encodes an antibody or fragment thereof comprising a heavy chain or VH described herein. In certain embodiments, the nucleotide sequence comprises a sequence that encodes the VH region of H1-21G6, H2-21G6, H3-21G6, or H4-21G6. Exemplary nucleotide sequences include SEQ ID NOs: 56, 57, 58, and 59 respectively (shown below). In some embodiments, the nucleotide sequence that encodes an antibody described herein comprises a sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, and 59. In some embodiments, the nucleotide sequence is SEQ ID NO: 59. In certain additional embodiments, the invention encompasses a nucleotide sequence that encodes an antibody of fragment thereof that comprises a nucleotide that encodes a light chain or VL described herein. Exemplary nucleotide sequences encoding the VL regions L1-21G6, L2-21G6 and L3-21G6 are SEQ ID NOs: 61, 62 and 63, respectively (shown below). In some embodiments, the nucleotide sequence that encodes an antibody described herein comprises a sequence selected from the group consisting of SEQ ID NOs: 61, 62 and 63. In yet additional embodiments, the nucleotide sequence that encodes an antibody of antigen-binding fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, and 59 and a sequence selected from the group consisting of SEQ ID NOs: 61, 62 and 63. In yet additional embodiments, the nucleotide sequence comprises SEQ ID NO: 59 and SEQ ID NO: 62.

[0122] Also encompassed herein are nucleotide sequences that encode antibody, VH and/or VL regions described herein and that are optimized for increased production in specific expression systems. An exemplary method for optimizing expression of mRNA and/or protein is the GENEOPTIMIZER.RTM. Process from LifeTechnologies (see, for example, http://www.lifetechnologies.com/us/en/home/life-science/cloning/gene-synt- hesis/geneart-gene-synthesis/geneoptimizer.html). As shown in FIGS. 3 to 5, SEQ ID NOs: 64, 65, 68 and 69 represent optimized nucleotide sequences for production of the H4-21G6 Vh and L2-21G6 VI regions in CHO cells. In some embodiments, the invention encompasses a nucleotide sequence of SEQ ID NO: 64. In additional embodiments, the invention encompasses a nucleotide sequence of SEQ ID NO: 65. In yet further embodiments, the nucleotide comprises SEQ ID NO: 64 and SEQ ID NO: 65.

[0123] Also encompassed is an expression vector comprising a nucleotide sequence that encodes an antibody or antigen-binding fragment, a VH region and/or a VL region of the invention and an isolated cell comprising said vector. The antibody or antigen-binding fragment, can be produced, for example, by culturing a cell comprising said expression vector, recovering the antibody or fragment thereof from the cultured cells or culture medium. In some embodiments, the antibody or antigen-binding fragment is produced by culturing CHO cells comprising the expression vectors encompassed herein. In certain additional embodiments, the vector comprises a nucleotide sequence comprising SEQ ID NO: 64 and/or SEQ ID NO: 65. In yet additional embodiments, the vector comprises a nucleotide sequence comprising SEQ ID NO: 64 and SEQ ID NO: 65. In yet further embodiments, the vector comprises a nucleotide sequence comprising SEQ ID NOs: 68 or 69. "Cells" or "host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0124] As described above, the antibody and antigen-binding fragment of the invention bind the N2 epitope and can therefore be used for treating a number of inflammatory diseases and conditions that are triggered by binding of natural IgM antibodies. For instance, the antibodies or fragments thereof can be used to treat inflammatory diseases or conditions such as reperfusion injury, ischemia injury, stroke, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, celiac disease, hyper-IgG immunodeficiency, arteriosclerosis, coronary artery disease, sepsis, myocarditis, encephalitis, transplant rejection, hepatitis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves disease), osteoporosis, polymyositis, dermatomyositis, drug- or chemotherapy-induced inflammation (e.g., drug or chemotherapy induced nephritis, endocarditis, nephritis), Type I diabetes, gout, dermatitis, alopecia areata, systemic lupus erythematosus, lichen sclerosis, ulcerative colitis, diabetic retinopathy, pelvic inflammatory disease, periodontal disease, arthritis, juvenile chronic arthritis (e.g., chronic iridocyclitis), psoriasis, osteoporosis, nephropathy in diabetes mellitus, asthma, pelvic inflammatory disease, chronic inflammatory liver disease, chronic inflammatory lung disease, lung fibrosis, liver fibrosis, rheumatoid arthritis, chronic inflammatory liver disease, chronic inflammatory lung disease, lung fibrosis, liver fibrosis, Crohn's disease, ulcerative colitis, burn injury (or thermal injury), and other acute and chronic inflammatory diseases of the Central Nervous System (CNS; e.g., multiple sclerosis), gastrointestinal system, the skin and associated structures, the immune system, the hepato-biliary system, or any site in the body where pathology can occur with an inflammatory component.

[0125] The invention encompasses methods of inhibiting the activation of an immune response to the N2 antigen in a subject by administering to a subject an antibody described herein. In a further aspect, the invention encompasses methods of treating an inflammatory disease or condition, for example, ischemia-reperfusion injury, in a subject comprising administering to the subject a pharmaceutical composition comprising an antibody or fragment of the invention.

[0126] An inflammatory condition such as reperfusion or ischemic injury can result following a naturally occurring episode, including, for example, a stroke or myocardial infarction. Reperfusion or ischemic injury can also occur during and/or following a surgical procedure. Exemplary surgical procedures that cause can cause injury include a vessel-corrective technique selected from the group consisting of angioplasty, stenting procedure, atherectomy, and bypass surgery. In an exemplary embodiment, reperfusion or ischemic injury occurs in a cardiovascular tissue, such as the heart.

[0127] Data obtained from animal studies can be used in formulating a range of dosage for use in humans. The dosage of an antibody is within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For an antibody or fragment thereof used in the method described herein, the therapeutically effective dose can be estimated initially from in vitro assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in in vitro assay. This information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, using enzyme linked immunosorbent assay (ELISA).

[0128] In some embodiments, an antibody or fragment thereof can be administered prior to, contemporaneously with, or subsequent to a tissue injury. In some embodiments, the pharmaceutical composition can be administered a few hours, a few days or a few weeks after tissue injury. In some embodiments, an antibody or fragment thereof can be administered prior to tissue injury, for example, in subjects at risk for reperfuion injury such as those patients that are about to undergo surgery. In additional embodiments, the antibody or fragment thereof can be administered.

[0129] A "therapeutically effective amount" or an "effective amount" is an amount which, alone or in combination with one or more other active agents, can control, decrease, inhibit, ameliorate, prevent or otherwise affect and/or achieve a recited effect. An effective amount of the agent to be administered can be determined using methods well-known in the art. One of skill in the art would take into account the mode of administration, the disease or condition (if any) being treated and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs. A "patient" can refer to a human subject in need of treatment.

[0130] The antibody or fragment of the present invention can be provided in pharmaceutically acceptable carriers or formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. In certain embodiments, the antibody or fragment thereof is provided for transmucosal or transdermal delivery. For such administration, penetrants appropriate to the barrier to be permeated are used in the formulation with the polypeptide. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, compositions of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

[0131] The pharmaceutical compositions according to the invention are prepared by bringing an antibody or fragment thereof into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include, for example, antimicrobial, anti-oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000) and Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences, 2012), and Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975) and The National Formulary XIV., 14th ed. Washington: American Pharmaceutical Association (1975), the contents of each of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed.) and Goodman and Gilman's The Pharmacological Basis for Therapeutics, 12th edition, (McGraw Hill Professional Publishing, 2010).

[0132] The pharmaceutical compositions can be prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories and including, for example, alginate based pH dependent release gel caps. For treatment of a subject, depending on activity of the pharmaceutical composition, the manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses can be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or by several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.

[0133] The pharmaceutical compositions according to the invention can be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. As discussed above, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Langer, Science, 249: 1527, (1990); Goodman and Gilman's The Pharmacological Basis for Therapeutics, 12th edition, (McGraw Hill Professional Publishing, 2010); each of which is herein incorporated by reference.

[0134] In one embodiment, the invention provides a pharmaceutical composition useful for administering an antibody or antigen-binding fragment thereof to a subject in need of such treatment. "Administering" the pharmaceutical composition of the invention may be accomplished by any means known to the skilled artisan. A "subject" refers to a mammal, most preferably a human.

[0135] The antibody or fragment thereof can be administered parenterally, enterically, by injection, rapid infusion, nasopharyngeal absorption, dermal absorption, rectally and orally. Pharmaceutically acceptable carrier preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers for occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners and elixirs containing inert diluents commonly used in the art, such as purified water. Where the disease or disorder is a gastrointestinal disorder oral formulations or suppository formulations are preferred.

[0136] Sterile injectable solutions can be prepared by incorporating an antibody or antigen-binding fragment thereof in the required amount (e.g., about 10 .mu.g to about 10 mg/kg) in an appropriate solvent and then sterilizing, such as by sterile filtration. Further, powders can be prepared by standard techniques such as freeze drying or vacuum drying.

[0137] In another embodiment, antibody or fragment thereof is prepared with a biodegradable carrier for sustained release characteristics for either sustained release in the GI tract or for target organ implantation with long term active agent release characteristics to the intended site of activity. Biodegradable polymers include, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acids, polylactic acids, collagen, polyorthoesters, and poly acetic acid. Liposomal formulation can also be used.

[0138] Any route of administration compatible with the active principle can be used. In some embodiments, the route of administration is parenteral administration, such as subcutaneous, intramuscular or intravenous injection. The dose of the antibody or antigen-binding fragment thereof to be administered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.

[0139] The daily non-weighted dosage for the patient can be between about 2.5-5.0 mg/Kg, e.g., about 2.5-3.0 mg/Kg, about 3.0-3.5 mg/Kg, about 3.5-4.0 mg/Kg, about 4.0-4.5 mg/Kg, and about 4.5-5.0 mg/Kg.

[0140] The pharmaceutical composition for parenteral administration can be prepared in an injectable form comprising the active principle and a suitable vehicle. Vehicles for the parenteral administration are well known in the art and comprise, for example, water, saline solution, Ringer solution and/or dextrose. The vehicle can contain small amounts of excipients in order to maintain the stability and isotonicity of the pharmaceutical preparation. The preparation of the cited solutions can be carried out according to the ordinary modalities.

[0141] The present invention has been described with reference to the specific embodiments, but the content of the description comprises all modifications and substitutions, including conservative amino acid substitutions, which can be brought by a person skilled in the art without extending beyond the meaning and purpose of the claims. The compositions can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0142] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, about 60%, and even more preferably at least about 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0143] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences and the percent homology between two sequences is a function of the number of conserved positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity and/or homology between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the internet at the Accelrys website, more specifically at http://www.accelrys.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available on the world wide web with the extension gcg.com), using a NWSgapdna CMP matrix and a gap weight of 40, 50, 60, 70; or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.

[0144] The percent identity and/or homology between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0145] "Stringency hybridization" or "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" is used herein to describe conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6:3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature of the washes can be increased to 55.degree. C. for low stringency conditions); 2) medium stringency hybridization conditions in 6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency hybridization conditions in 6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by one or more washes at 0.2.times.SSC, 1% SDS at 65.degree. C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified. Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as described above.

[0146] The amino acid sequences referred to in the present application are listed below with the corresponding sequence identifier (SEQ ID NO):

TABLE-US-00012 m21G6 VH (SEQ ID NO: 1) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGG INPSNGGTNFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRWG YDREWFAYWGQGTLVTVSA. m21G6 VL (SEQ ID NO: 2) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQ VLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYP FTFGSGTKLEIKR. VH CDR1 (SEQ ID NO: 3) SYYMY. VH CDR2 (SEQ ID NO: 4) GINPSNGGTNFNEKFKS. VH CDR3 (SEQ ID NO: 5) GYDREWFAY. VL CDR1 (SEQ ID NO: 6) RSSKSLLHSNGNTYLY. VL CDR2 (SEQ ID NO: 7) RMSNLAS. VL CDR3 (SEQ ID NO: 8) MQHLEYPFT. H1-21G6 Vh (SEQ ID NO: 9) QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYYMYWVKQAPGQGLEWIGG INPSNGGTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRWG YDREWFAYWGQGTLVTVSS. H2-21G6 Vh (SEQ ID NO: 10) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKSKATMTVDKSTSTAYMELRSLRSDDSAVYYCTRWG YDREWFAYWGQGTLVTVSS. H3-21G6 Vh (SEQ ID NO: 11) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKSKATITVDKSTSTAYMELSSLRSEDTAVYYCTRWG YDREWFAYWGQGTLVTVSS. H4-21G6 Vh (SEQ ID NO: 49) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKSRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRWG YDREWFAYWGQGTLVTVSS. L1-21G6 Vl (PopVk, CLL) (SEQ ID NO: 12) DIVMTQSPATLSVSPGERATISCRSSKSLLHSNGNTYLYWFQQKPGQPPK VLIYRMSNLASGVPARFSGSGSGTDFTLTISSVEPEDFATYYCMQHLEYP FTFGGGTKLEIKR. L2-21G6 Vl (SEQ ID NO: 13) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYP FTFGQGTKLEIKR. L3-m21G6 Vl (SEQ ID NO: 14) DIVMTQTPLSLSYTPGQPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYP FTFGQGTKLEIKR. H1 VH FWR1 (SEQ ID NO: 15) QVQLVQSGAEVVKPGASVKLSCKASGYTFT. H1 VH FWR2 (SEQ ID NO: 16) WVKQAPGQGLEWIG. H1 VH FWR3 (SEQ ID NO: 17) KATLTVDKSASTAYMELSSLRSEDTAVYYCTR. H1 VH FWR4 (SEQ ID NO: 18) WGQGTLVTVSS. H2 VH FWR1 (SEQ ID NO: 19) QVQLVQSGAEVKKPGASVKVSCKASGYTFT. H2 VH FWR2 (SEQ ID NO: 20) WVRQAPGQGLEWIG. H2 VH FWR3 (SEQ ID NO: 21) KATMTVDKSTSTAYMELRSLRSDDSAVYYCTR. H2 VH FWR4 (SEQ ID NO: 22) WGQGTLVTVSS. H3 VH FWR1 (SEQ ID NO: 23) QVQLVQSGAEVKKPGSSVKVSCKASGYTFT. H3 VH FWR2 (SEQ ID NO: 24) WVRQAPGQGLEWIG. H3 VH FWR3 (SEQ ID NO: 25) KATITVDKSTSTAYMELSSLRSEDTAVYYCTR. H3 VH FWR4 (SEQ ID NO: 26) WGQGTLVTVSS. H4 VH FWR1 (SEQ ID NO: 19) QVQLVQSGAEVKKPGASVKVSCKASGYTFT H4 VH FWR2 (SEQ ID NO: 50) WVRQAPGQGLEWMG. H4 VH FWR3 (SEQ ID NO: 51) RVTMTTDTSTSTAYMELRSLRSDDTAVYYCTR. H4 VH FWR4 (SEQ ID NO: 22) WGQGTLVTVSS. L1 VL FWR1 (SEQ ID NO: 27) DIVMTQSPATLSVSPGERATISC. L1 VL FWR2 (SEQ ID NO: 28) WFQQKPGQPPKVLIY. L1 VL FWR3 (SEQ ID NO: 29) GVPARFSGSGSGTDFTLTISSVEPEDFATYYC. L1 VL FWR4 (SEQ ID NO: 30) FGGGTKLEIKR. L2 VL FWR1 (SEQ ID NO: 31) DIVMTQSPLSLPVTPGEPASISC. L2 VL FWR2 (SEQ ID NO: 32) WFLQKPGQSPQLLIY. L2 VL FWR3 (SEQ ID NO: 33) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC. L2 VL FWR4 (SEQ ID NO: 34) FGQGTKLEIKR. L3 VL FWR1 (SEQ ID NO: 35) DIVMTQTPLSLSYTPGQPASISC. L3 VL FWR2 (SEQ ID NO: 36) WFLQKPGQSPQLLIY. L3 VL FWR3 (SEQ ID NO: 37) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC. L3 VL FWR4 (SEQ ID NO: 38) FGQGTKLEIKR. H1 VH FWR3 with amino acid mutation to alanine (SEQ ID NO: 39) KATLTVDKSASTAYMELSSLRSEDTAVYYCAR. H2 VH FWR3 with amino acid mutation to alanine (SEQ ID NO: 40) KATMTVDKSTSTAYMELRSLRSDDSAVYYCAR. H3 VH FWR3 with amino acid mutation to alanine (SEQ ID NO: 41) KATITVDKSTSTAYMELSSLRSEDTAVYYCAR. H4 VH FWR3 (SEQ ID NO: 52) RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR Amino Acid Variant of H2 Vh (SEQ ID NO: 42) QVQLVQSGAEVKKPGASLKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKGRVTITRDKSTSTAYMELRSLRSEDSAVYYCARWG YDREWFAYWGQGTLVTVSS. H1 VH with amino acid mutation to alanine in FWR3 (SEQ ID NO: 43) QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYYMYWVKQAPGQGLEWIGG INPSNGGTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCARWG YDREWFAYWGQGTLVTVSS. H2 VH with amino acid mutation to alanine in FWR3 (SEQ ID NO: 44) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKSKATMTVDKSTSTAYMELRSLRSDDSAVYYCARWG YDREWFAYWGQGTLVTVSS. H3 with amino acid mutation to alanine in FWR3 (SEQ ID NO: 45) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGG INPSNGGTNFNEKFKSKATITVDKSTSTAYMELSSLRSEDTAVYYCARWG YDREWFAYWGQGTLVTVSS. H4 VH with amino acid mutation to alanine in FWR3 (SEQ ID NO: 53) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKSRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWG YDREWFAYWGQGTLVTVSS. Amino Acid Variant of kappa chain (m21G6 Vl) (SEQ ID NO: 46) EIVLTQSPGTLSLSPGERATLSCRASKSLLHSNGNTYLYWYQQKPGQAPR LLIYRMSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCMQHLEYP FTFGQGTKLEIKR. N2 peptide (SEQ ID NO: 47) LMKNMDPLNDNV. Peptide sequence (SEQ ID NO: 48) LMKNMDPLNDNI.

H4-21G6 Vh with leader sequence and the constant region of human IgG1 (SEQ ID NO: 54) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTS YYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKSRVTMTTDTSTSTAYM ELRSLRSDDTAVYYCTRWGYDREWFAYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK. L2-21G6 Vl with leader sequence and the constant region of the human kappa light chain (SEQ ID NO: 55) MGWSCIILFLVATATGVHGDIVMTQSPLSLPVTPGEPASISCRSSKSLLH SNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKIS RVEAEDVGVYYCMQHLEYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. Nucleotide sequence encoding H1-21G6 Vh (SEQ ID NO: 56) CAGGTCCAACTGGTGCAGtCTGGGGCTGAAGTGGTGAAGCCTGGGGCTTC AGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATA TGTACTGGGTGAAGCAGGCGCCTGGACAAGGCCTTGAGTGGATTGGGGGG ATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGAGCAA GGCCACACTGACTGTAGACAAATCCGCCAGCACAGCCTACATGGAACTCA GCAGCCTGAGATCTGAGGACACTGCGGTCTATTACTGTACAAGATGGGGT TACGACAGGGAGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTTCA. Nucleotide sequence encoding H2-21G6 Vh (SEQ ID NO: 57) CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCTTC AGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATA TGTACTGGGTGAGGCAGGCGCCTGGACAAGGCCTTGAGTGGATTGGGGGG ATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGAGCAA GGCCACAATGACTGTAGACAAATCCACCAGCACAGCCTACATGGAACTCC GCAGCCTGAGATCTGACGACACTGCGGTCTATTACTGTACAAGATGGGGT TACGACAGGGAGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTTCA. Nucleotide sequence encoding H3-21G6 Vh (SEQ ID NO: 58) CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCTTC AGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATA TGTACTGGGTGAGGCAGGCGCCTGGACAAGGCCTTGAGTGGATTGGGGGG ATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGAGCAA GGCCACAATCACTGTAGACAAATCCACCAGCACAGCCTACATGGAACTCA GCAGcCTGAGATCTGAGGACACTGCGGTCTATTACTGTACAAGATGGGGT TACGACAGGGAGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTTCA. Nucloetide sequence encoding H4-21G6 Vh (SEQ ID NO: 59) CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCTTC AGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATA TGTACTGGGTGAGGCAGGCGCCTGGACAAGGCCTTGAGTGGATGGGGGGG ATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCAAGAGCAG GGTCACAATGACTACAGACACATCCACCAGCACAGCCTACATGGAACTCC GCAGCCTGAGATCTGACGACACTGCGGTCTATTACTGTACAAGATGGGGT TACGACAGGGAGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTTCA. Optimized nucleotide sequence encoding H4-21G6 Vh including intron from vector, leader sequence and IgG1 constant region (SEQ ID NO: 60) CACTATAGGGCGAATTGAAGGAAGGCCGTCAAGGCCGCATCCTAGGGCCA CCATGGGCTGGTCCTGCATCATCCTGTTTCTGGTCGCCACCGCCACCGGC GTGCACTCCCAGGTCCAGCTGGTCCAGTCTGGCGCCGAAGTGAAGAAACC TGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCA GCTACTACATGTACTGGGTCCGACAGGCCCCAGGCCAGGGACTGGAATGG ATGGGCGGCATCAACCCCTCCAACGGCGGCACCAACTTCAACGAGAAGTT CAAGTCCAGAGTGACCATGACCACCGACACCTCCACCTCCACCGCCTACA TGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCACC AGATGGGGCTACGACAGAGAGTGGTTCGCCTACTGGGGCCAGGGCACCCT GGTCACAGTGTCCTCCGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGG CCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCCCTGGGCTGCCTG GTCAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGC CCTGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCTTCCGGCC TGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCCAGCTCTCTGGGCACC CAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGA CAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCT GCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCA AAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGT GGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACG TGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAG TACCAGTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGC CTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAG CCCCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACCAAGAACCA GGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGACATTGCCG TGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCC CCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGT GGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGC ACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCC GGCAAGTGATGAGTATACCTGGGCCTCATGGGCCTTCCTTTCACTGCCCG CTTTCCAG. Nucleotide sequence encoding L1-21G6 Vl (SEQ ID NO: 61) GATATTGTGATGACTCAGTCTCCAGCCACTCTATCTGTCTCTCCTGGAGA GCGAGCAACCATCTCCTGCAGGGCTAGTAAGAGTCTCCTGCATAGTAATG GCAACACTTACTTGTATTGGTTCCAGCAGAAGCCAGGCCAGCCTCCTAAG GTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGCCAGGTT CAGTGGCAGTGGGTCAGGAACTGATTTCACACTGACAATCAGTTCggtgG AGccTGAGGATTTTGCTACTTATTACTGTATGCAACATCTAGAATATCCA TTCACGTTCGGCGGGGGGACAAAGTTGGAAATAAAACG. Nucleotide sequence encoding L2-21G6 Vl (SEQ ID NO: 62) GATATTGTGATGACTCAGTCTCCACTCTCTCTACCTGTCACTCCTGGAGA GcCAGCATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATG GCAACACTTACTTGTATTGGTTCCTGCAGAAGCCAGGCCAGTCTCCTCAG cTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTT CAGTGGCAGTGGGTCAGGAACTGcTTTCACACTGAAAATCAGTAGAGTGG AGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCA TTCACGTTCGGCCAGGGGACAAAGCTGGAAATAAAACG. Nucleotide sequence encoding L3-21G6 Vl (SEQ ID NO: 63) GATATTGTGATGACTCAGACTCCACTCTCTCTAtCTGTCACTCCTGGAcA GcCAGCATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATG GCAACACTTACTTGTATTGGTTCCTGCAGAAGCCAGGCCAGTCTCCTCAG cTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTT CAGTGGCAGTGGGTCAGGAACTGaTTTCACACTGAAAATCAGTAGAGTGG AGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCA TTCACGTTCGGCCAGGGGACAAAGCTGGAAATAAAACG. Optimized nucleotide sequence encoding H4-21G6 Vh (SEQ ID NO: 64) CAGGTCCAGCTGGTCCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTC CGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAGCTACTACA TGTACTGGGTCCGACAGGCCCCAGGCCAGGGACTGGAATGGATGGGCGGC ATCAACCCCTCCAACGGCGGCACCAACTTCAACGAGAAGTTCAAGTCCAG AGTGACCATGACCACCGACACCTCCACCTCCACCGCCTACATGGAACTGC GGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCACCAGATGGGGC TACGACAGAGAGTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTCACAGT GTCCTCC. Optimized nucleotide sequence encoding L2-21G6 Vl (SEQ ID NO: 65) GACATCGTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACACCTGGCGA GCCTGCCTCCATCTCCTGCCGGTCCTCCAAGTCCCTGCTGCACTCCAACG GCAATACCTACCTGTACTGGTTCCTGCAGAAGCCCGGCCAGTCCCCTCAG CTGCTGATCTACCGGATGTCCAACCTGGCCTCCGGCGTGCCCGACAGATT CTCCGGCTCTGGCTCTGGCACAGCCTTCACCCTGAAGATCTCCCGGGTGG

AAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAATACCCC TTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGG. Nucleotide sequence encoding H4-21G6 Vh including leader sequence and constant region (SEQ ID NO: 66) ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGT CCACTCCCAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTG GGGCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGC TACTATATGTACTGGGTGAGGCAGGCGCCTGGACAAGGCCTTGAGTGGAT GGGGGGGATTAATCCTAGCAATGGTGGTACTAACTTCAATGAGAAGTTCA AGAGCAGGGTCACAATGACTACAGACACATCCACCAGCACAGCCTACATG GAACTCCGCAGCCTGAGATCTGACGACACTGCGGTCTATTACTGTACAAG ATGGGGTTACGACAGGGAGTGGTTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCA CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT CAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGG TAAATGA. Nucleotide sequence encoding L2-21G6 Vl including leader sequence and human kappa light chain (SEQ ID NO: 67) ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGT ACATGGGGATATTGTGATGACTCAGTCTCCACTCTCTCTACCTGTCACTC CTGGAGAGCCAGCATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCAT AGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAAGCCAGGCCAGTC TCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAG ACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAAAATCAGT AGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGA ATATCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATAAAACGTACGG TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA ACAGGGGAGAGTGTTAG. Optimized nucleotide sequence encoding H4-21G6 Vh with leader sequence and IgG1 constant region (SEQ ID NO: 68) ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTCGCCACCGCCACCGGCGT GCACTCCCAGGTCCAGCTGGTCCAGTCTGGCGCCGAAGTGAAGAAACCTG GCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAGC TACTACATGTACTGGGTCCGACAGGCCCCAGGCCAGGGACTGGAATGGAT GGGCGGCATCAACCCCTCCAACGGCGGCACCAACTTCAACGAGAAGTTCA AGTCCAGAGTGACCATGACCACCGACACCTCCACCTCCACCGCCTACATG GAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCACCAG ATGGGGCTACGACAGAGAGTGGTTCGCCTACTGGGGCCAGGGCACCCTGG TCACAGTGTCCTCCGCTTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCC CCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCCCTGGGCTGCCTGGT CAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCC TGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCTTCCGGCCTG TACTCCCTGTCCTCCGTGGTCACCGTGCCCTCCAGCTCTCTGGGCACCCA GACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACA AGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGC CCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAA GCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGG TGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTG GACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTA CCAGTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACT GGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCT GCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCC CCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACCAAGAACCAGG TGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGACATTGCCGTG GAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCC TGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGG ACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCAC GAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGG CAAGTGATGAGTATACCTGGGCCTCATGGGCCTTCCTTTCACTGCCCGCT TTCCAG. Optimized nucleotide sequence encoding L2-21G6 Vl including leader sequence and human kappa light chain (SEQ ID NO: 69) CGAATTGGCGGAAGGCCGTCAAGGCCACGTGTCTTGTCCAGAGCTCGATA TCGCCACCATGGGCTGGTCCTGCATCATCCTGTTTCTGGTCGCCACCGCC ACCGGCGTGCACGGCGACATCGTGATGACCCAGTCCCCCCTGTCCCTGCC CGTGACACCTGGCGAGCCTGCCTCCATCTCCTGCCGGTCCTCCAAGTCCC TGCTGCACTCCAACGGCAATACCTACCTGTACTGGTTCCTGCAGAAGCCC GGCCAGTCCCCTCAGCTGCTGATCTACCGGATGTCCAACCTGGCCTCCGG CGTGCCCGACAGATTCTCCGGCTCTGGCTCTGGCACAGCCTTCACCCTGA AGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAG CACCTGGAATACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAA GCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGC AGCTGAAGTCCGGCACCGCCTCCGTCGTCTGCCTGCTGAACAACTTCTAC CCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGG CAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACT CCCTGTCTTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAG GTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA GTCCTTCAACCGGGGCGAGTGCTGATGATTAATTAAGGTACCTGGAGCAC AAGACTGGCCTCATGGGCCTTCCGCTCACTGC.

[0147] The invention is illustrated by the following non-limiting examples.

EXEMPLIFICATION

Example 1

Humanization of Murine Antibody 21G6

[0148] Murine 21G6 is an IgG1 heavy chain and kappa light chain that was raised against the non-muscle myosin neo-epitope N2 12mer sequence: LMKNMDPLNDNV (SEQ ID NO: 47). The murine 21G6 antibody is described in more detail in U.S. Pat. No. 8,324,352, the contents of which are expressly incorporated herein. Using the IMGT database (http://www.imgt.org), a search was performed to identify the human germline antibody sequences with the greatest homology to the murine 21G6 antibody. In addition, a BLAST search was performed to identify homologous human non-germline antibodies. The sequences shown in FIG. 1 were determined to have the highest amino acid homology.

[0149] FIG. 1 shows a sequence comparison for the murine 21G6 heavy chain variable (VH) region variable heavy and the humanized heavy chain variable regions (VH) H1-21G6, H2-21G6 and H3-21G6 and also shows a sequence comparison of the murine 21G6 light chain variable (VL) region and the humanized light chain variable regions light chain (VL) regions L1-21G6, L2-21G6 and L3-21G6.

[0150] The H1-21G6 and L1-21G6 frameworks were derived from B-cells obtained from lupus and chronic lymphocytic leukemia (CLL) patients for the heavy and light chains, respectively. The remaining sequences represent the germline sequences with the highest homology that encode productive antibody. All of the humanized sequences maintain the murine 21G6 CDR regions which are shown inside the boxes in FIG. 1. The humanized variable regions were cloned into a vector containing wild type human IgG1, human IgG1 containing a mutation at amino acid 297 (Asn 297 to Q297), human IgG4 containing a mutation at amino acid 228 (serine to proline) and human kappa light chain. Each antibody (heavy and light) combination was expressed by transient co-transfection in 293A cells (in the presence of low Ig serum). The antibody containing supernatants were collected and analyzed for binding to the N2 peptide by ELISA and BIACORE.TM. X100 system. All experiments were performed using a BIACORE.TM. X100 system. For antibody capture experiments, a CM5 chip was prepared by 10 ul/minute injection of EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) for 7 minutes, followed by a 10 ul/minute injection of anti-human Fc (GE Lifesciences) at a concentration of 25 ug/ml in sodium acetate at pH 5 for 3 minutes. Ethanolamine-HCl was injected for 7 minutes at 10 ul/minute. The chimeric or humanized antibodies were captured onto flow cell 2 and N2 peptide at varying concentrations was flowed over flow cells 1 and 2 at a rate of 30 ul/minute with a contact time of 120 seconds and a dissociation period of 120 seconds. Complete removal of captured antibody was accomplished by regeneration with 3M MgCl2 for 30 seconds at a flow rate of 10 ul/minute. For peptide immobilized experiments, a CM5 chip was prepared by 10 ul/minute injection of EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) for 7 minutes, followed by a 10 ul/minute injection of NEUTRAVIDIN.RTM. protein (ThermoFisher Pierce) at a concentration of 5 ug/ml in sodium acetate, pH 5 to an immobilization response of 100-200RU. Ethanolamine-HCl was injected for 7 minutes at 10 ul/minute. Biotin-labeled N2 peptide was captured on flow channel 2 with the goal of an experimental Rmax of about 50-100RU. Purified chimeric or humanized antibodies were flowed over both flow channels at a rate of 30 ul/min for a contact time of 120 seconds and allowed to dissociate for 600 seconds. Regeneration was achieved with glycine pH 1.7 for 30 seconds thus retaining an active surface. The apparent affinity constants and antibody on/off rates are shown in Tables 1A, 1B, 2, 3A and 3B below:

TABLE-US-00013 TABLE 1A Apparent Affinity Constants for variable regions and wild type human IgG1 heavy chain (Immobilized Antibody, N2 peptide in solution) Kd (uM) Heavy Light by IgG Chain Chain capture H1 L2 17 H1 L3 20.9 H1 L1 6.6-7.7 H2 L2 6.4-8.4 H2 L3 9.56 H2 L1 9.8-11 H3 L2 11.5 H3 L3 9.38 H3 L1 7.9 m21G6 m21G6 4-6 CHIgG1 Ch 6-7 (human heavy chain constant region IgG1) CHIgG4 Ch 4.1 (human heavy chain constant region IgG4)

TABLE-US-00014 TABLE 1B Apparent Affinity Constants for variable regions and human IgG1 heavy chain with mutation at position 297 from Asn to Gln (Immobilized Antibody, N2 peptide in solution) Heavy Kd (uM) Chain Light by IgG N297Q Chain capture H1 L2 TBD H1 L3 TBD H1 L1 9 H2 L2 3.1-3.7 H2 L3 8.8 H2 L1 TBD H3 L2 TBD H3 L1 20 H3 L3 TBD CHIgG1 CH 5.6 (human heavy chain constant region IgG1) (TBD indicated "to be determined")

TABLE-US-00015 TABLE 2 Antibody on- and off-rates (immobilized N2, antibody in solution) Steady Affinity (nM) state Heavy Light Ka (e + 4) kd (e - 3) kd/ka affinity Chain Chain (on-rate) (off-rate) (monovalent) (nM) H1 L3 0.63 9.93 1580 1000 H1 L1 1.5-4.3 4.3-6.5 100-430 320 H2 L2 2.0-3.2 8.6-9.1 280-420 240 H2 L3 2.4 3.5 140 680 H2 L1 0.7 4.63 660 NA H3 L2 1.2 7.2 640 NA H3 L3 0.60 5.61 900 NA H3 L1 2.0-5.2 3-6.5 300 280 ch21G6 ch21G6 7-10 7-19 100-200 196-240 N297Q (human heavy chain constant region IgG1 with Q297 mutation) ch21G6 IgG4 ch21G6 5 40 800 113 (human heavy chain constant region IgG4) m21G6 m21G6 1-11 5-53 200-400 313-540

TABLE-US-00016 TABLE 3A Apparent Affinity Constant for variable region H4 and IgG1 heavy chain with mutation at position 297 from Asn to Gln (Immobilized Antibody, N2 peptidein solution) Kd (uM) by Heavy IgG capture Chain Light and direct (N297Q) Chain immobilization H4 L2 6-11

TABLE-US-00017 TABLE 3B Antibody on- and off-rates (immobilized N2, antibody in solution) Steady Affinity (nM) state Heavy Light Ka (e + 4) kd (e - 3) kd/ka affinity Chain Chain (on-rate) (off-rate) (monovalent) (nM) H4 L2 2 2 100 TBD

[0151] As determined by t-test, there was no significant difference between murine 21G6 and H1/L1 and murine 21G6 and H2/L2. (Murine 21G6: n=4; 9r/9r: n=3; 69/9r: n=2).

Example 2

Optimization of Nucleotide Sequences for Increased Production in CHO Cells

[0152] Optimization was performed in order to use codon preferences of Cricetulus griseus (Chinese hamster) for optimal expression of the humanized antibody. The GENEOPTIMIZER.RTM. program also optimized sequence to prevent aberrant mRNA splicing, eliminate undesirable polyA binding motifs, optimize GC content of the gene and prevent unwanted secondary RNA structures that might decrease protein translation efficiency, thereby increasing overall antibody expression. Amino acid and nucleotides sequences for H4-21G6 and L2-21G6 (SEQ ID NOs: 54, 66, 55 and 67, respectively) were sent to Life Technologies for optimization using the GENEOPTIMIZER.RTM. Process described at http://www.lifetechnologies.com/us/en/home/life-science/cloning/gene-synt- hesis/geneart-gene-synthesis/geneoptimizer.html (the contents of which are expressly incorporated by reference herein). FIG. 3 shows a nucleotide sequence encoding the humanized heavy chain variable region H4 that was optimized for the production of the humanized antibody in Chinese hamster ovary (CHO) cells where each + indicates where a change was made as compared to SEQ ID NO: 66. FIG. 4 shows a nucleotide sequence encoding the humanized light chain variable region L2 that was optimized for the production of the humanized antibody in Chinese hamster ovary (CHO) cells. Each + indicates where a change was made as compared to SEQ ID NO: 67. FIG. 5 depicts the levels of recombinant antibody expression on day 5 post-transfection with either native (red) or optimized (blue) antibody sequences. Incorporation of the recombinant antibody sequence into the CHO cells was accomplished by selection in the presence of 10 ug/ml puromycin and 100 mM methotrexate for transfectants DS5 and DS7 or 20 ug/ml puromycin and 200 mM methotrexate for transfectants DS6 and DS8.

[0153] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Sequence CWU 1

1

691119PRTMus sp. 1Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 2113PRTMus sp. 2Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 35PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 3Ser Tyr Tyr Met Tyr 1 5 417PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Ser 59PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 5Gly Tyr Asp Arg Glu Trp Phe Ala Tyr 1 5 616PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 77PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Arg Met Ser Asn Leu Ala Ser 1 5 89PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 8Met Gln His Leu Glu Tyr Pro Phe Thr 1 5 9119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 9Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 10119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 10Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 11119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 11Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 12113PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 12Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 13113PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 13Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 14113PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 14Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Tyr Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 1530PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 1614PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 16Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 1732PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 17Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 1811PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 18Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 1930PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 19Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 2014PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 20Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 2132PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 21Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Arg Ser Leu Arg Ser Asp Asp Ser Ala Val Tyr Tyr Cys Thr Arg 20 25 30 2211PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 22Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 2330PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 2414PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 24Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 2532PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 25Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 2611PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 26Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 2723PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 27Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys 20 2815PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 28Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr 1 5 10 15 2932PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 29Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 3011PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 30Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 3123PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 31Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys 20 3215PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 32Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 3332PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 33Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 3411PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 34Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 3523PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 35Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Tyr Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 3615PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 36Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 3732PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 37Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 3811PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 3932PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 39Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 4032PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 40Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Arg Ser Leu Arg Ser Asp Asp Ser Ala Val Tyr Tyr Cys Ala Arg 20 25 30 4132PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 41Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 42119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 43119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 43Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly

Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 44119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 44Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 45119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 45Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 46113PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 46Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Arg Met Ser Asn Arg Ala Thr Gly Ile Pro 50 55 60 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 4712PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 47Leu Met Lys Asn Met Asp Pro Leu Asn Asp Asn Val 1 5 10 4812PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 48Leu Met Lys Asn Met Asp Pro Leu Asn Asp Asn Ile 1 5 10 49119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 49Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 5014PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 50Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 5132PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 51Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 5232PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 52Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 53119PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 53Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 54468PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 54Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn 65 70 75 80 Glu Lys Phe Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 55238PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 55Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25 30 Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu 35 40 45 Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 100 105 110 Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 56357DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 56caggtccaac tggtgcagtc tggggctgaa gtggtgaagc ctggggcttc agtgaagttg 60tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaagcaggcg 120cctggacaag gccttgagtg gattgggggg attaatccta gcaatggtgg tactaacttc 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aatccgccag cacagcctac 240atggaactca gcagcctgag atctgaggac actgcggtct attactgtac aagatggggt 300tacgacaggg agtggtttgc ttactggggc caagggactc tggtcactgt ctcttca 35757357DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 57caggtccaac tggtgcagtc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaggcaggcg 120cctggacaag gccttgagtg gattgggggg attaatccta gcaatggtgg tactaacttc 180aatgagaagt tcaagagcaa ggccacaatg actgtagaca aatccaccag cacagcctac 240atggaactcc gcagcctgag atctgacgac actgcggtct attactgtac aagatggggt 300tacgacaggg agtggtttgc ttactggggc caagggactc tggtcactgt ctcttca 35758357DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 58caggtccaac tggtgcagtc tggggctgaa gtgaagaagc ctgggtcttc agtgaaggtg 60tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaggcaggcg 120cctggacaag gccttgagtg gattgggggg attaatccta gcaatggtgg tactaacttc 180aatgagaagt tcaagagcaa ggccacaatc actgtagaca aatccaccag cacagcctac 240atggaactca gcagcctgag atctgaggac actgcggtct attactgtac aagatggggt 300tacgacaggg agtggtttgc ttactggggc caagggactc tggtcactgt ctcttca 35759357DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 59caggtccaac tggtgcagtc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaggcaggcg 120cctggacaag gccttgagtg gatggggggg attaatccta gcaatggtgg tactaacttc 180aatgagaagt tcaagagcag ggtcacaatg actacagaca catccaccag cacagcctac 240atggaactcc gcagcctgag atctgacgac actgcggtct attactgtac aagatggggt 300tacgacaggg agtggtttgc ttactggggc caagggactc tggtcactgt ctcttca 357601508DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 60cactataggg cgaattgaag gaaggccgtc aaggccgcat cctagggcca cc atg ggc 58 Met Gly 1 tgg tcc tgc atc atc ctg ttt ctg gtc gcc acc gcc acc ggc gtg cac 106Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His 5 10 15 tcc cag gtc cag ctg gtc cag tct ggc gcc gaa gtg aag aaa cct ggc 154Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 20 25 30 gcc tcc gtg aag gtg tcc tgc aag gcc tcc ggc tac acc ttc acc agc 202Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 35 40 45 50 tac tac atg tac tgg gtc cga cag gcc cca ggc cag gga ctg gaa tgg 250Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 55 60 65 atg ggc ggc atc aac ccc tcc aac ggc ggc acc aac ttc aac gag aag 298Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys 70 75 80 ttc aag tcc aga gtg acc atg acc acc gac acc tcc acc tcc acc gcc 346Phe Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala 85 90 95 tac atg gaa ctg cgg tcc ctg cgg agc gac gac acc gcc gtg tac tac 394Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr 100 105 110 tgc acc aga tgg ggc tac gac aga gag tgg ttc gcc tac tgg ggc cag 442Cys Thr Arg Trp Gly Tyr Asp Arg Glu Trp Phe Ala Tyr Trp Gly Gln 115 120 125 130 ggc acc ctg gtc aca gtg tcc tcc gct tcc acc aag ggc ccc tcc gtg 490Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 135 140 145 ttc cct ctg gcc ccc tcc agc aag tcc acc tct ggc ggc acc gct gcc 538Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 150 155 160 ctg ggc tgc ctg gtc aaa gac tac ttc ccc gag ccc gtg acc gtg tcc 586Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 tgg aac tct ggc gcc ctg acc agc ggc gtg cac acc ttc cct gcc gtg 634Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 ctg cag tct tcc ggc ctg tac tcc ctg tcc tcc gtg gtc acc gtg ccc 682Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 210 tcc agc tct ctg ggc acc cag acc tac atc tgc aac gtg aac cac aag 730Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 215 220 225 ccc tcc aac acc aag gtg gac aag cgg gtg gaa ccc aag tcc tgc gac 778Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 230 235 240 aag acc cac acc tgt ccc ccc tgc cct gcc cct gaa ctg ctg ggc gga 826Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255 cct tcc gtg ttc ctg ttc ccc cca aag ccc aag gac acc ctg atg atc 874Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 tcc cgg acc ccc gaa gtg acc tgc gtg gtg gtg gac gtg tcc cac gag 922Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 290 gac cct gaa gtg aag ttc aat tgg tac gtg gac ggc gtg gaa gtg cac 970Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 295 300 305 aac gcc aag acc aag ccc aga gag gaa cag tac cag tcc acc tac cgg 1018Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg 310 315 320 gtg gtg tct gtg ctg acc gtg ctg cac cag gac tgg ctg aac ggc aaa 1066Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 gag tac aag tgc aag gtc tcc aac aag gcc ctg cct gcc ccc atc gaa 1114Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 aag acc atc tcc aag gcc aag ggc cag ccc cgc gag ccc cag gtg tac 1162Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 370 aca ctg ccc cct agc cgg gaa gag atg acc aag aac cag gtg tcc ctg 1210Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 375 380 385 acc tgt ctg gtc aaa ggc ttc tac ccc tcc gac att gcc gtg gaa tgg 1258Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 390 395 400 gag tcc aac ggc cag ccc gag aac aac tac aag acc acc ccc cct gtg 1306Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 ctg gac tcc gac ggc tca ttc ttc ctg tac tcc aag ctg acc gtg gac 1354Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 aag tcc cgg tgg cag cag ggc aac gtg ttc tcc tgc tcc gtg atg cac 1402Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 450 gag gcc ctg cac aac cac tac acc cag aag tcc ctg tcc ctg agc ccc 1450Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 455 460 465 ggc aag tgatgagtat acctgggcct catgggcctt cctttcactg cccgctttcc ag 1508Gly Lys 61338DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 61gatattgtga tgactcagtc tccagccact ctatctgtct ctcctggaga gcgagcaacc 60atctcctgca gggctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 120ttccagcaga agccaggcca gcctcctaag gtcctgatat atcggatgtc caaccttgcc 180tcaggagtcc cagccaggtt cagtggcagt gggtcaggaa ctgatttcac actgacaatc 240agttcggtgg agcctgagga ttttgctact tattactgta tgcaacatct agaatatcca 300ttcacgttcg gcggggggac aaagttggaa ataaaacg 33862338DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 62gatattgtga tgactcagtc tccactctct ctacctgtca ctcctggaga gccagcatcc 60atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 120ttcctgcaga agccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 180tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgaaaatc 240agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcca 300ttcacgttcg gccaggggac aaagctggaa ataaaacg 33863338DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 63gatattgtga tgactcagac tccactctct ctatctgtca ctcctggaca gccagcatcc 60atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 120ttcctgcaga agccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 180tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgatttcac actgaaaatc 240agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcca 300ttcacgttcg gccaggggac aaagctggaa ataaaacg 33864357DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 64caggtccagc tggtccagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg 60tcctgcaagg cctccggcta caccttcacc agctactaca tgtactgggt ccgacaggcc 120ccaggccagg gactggaatg gatgggcggc atcaacccct ccaacggcgg caccaacttc 180aacgagaagt tcaagtccag agtgaccatg accaccgaca cctccacctc caccgcctac 240atggaactgc ggtccctgcg gagcgacgac accgccgtgt actactgcac cagatggggc 300tacgacagag agtggttcgc ctactggggc cagggcaccc tggtcacagt gtcctcc 35765339DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 65gacatcgtga tgacccagtc ccccctgtcc ctgcccgtga cacctggcga gcctgcctcc 60atctcctgcc ggtcctccaa gtccctgctg cactccaacg gcaataccta cctgtactgg 120ttcctgcaga agcccggcca gtcccctcag ctgctgatct accggatgtc caacctggcc 180tccggcgtgc ccgacagatt ctccggctct ggctctggca cagccttcac cctgaagatc 240tcccgggtgg aagccgagga cgtgggcgtg tactactgca tgcagcacct ggaatacccc 300ttcaccttcg gccagggcac caagctggaa atcaagcgg 339661407DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 66atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt ccactcccag 60gtccaactgg tgcagtctgg ggctgaagtg aagaagcctg gggcttcagt gaaggtgtcc 120tgcaaggctt ctggctacac cttcaccagc tactatatgt actgggtgag gcaggcgcct 180ggacaaggcc ttgagtggat gggggggatt aatcctagca atggtggtac taacttcaat 240gagaagttca agagcagggt cacaatgact acagacacat ccaccagcac agcctacatg 300gaactccgca gcctgagatc tgacgacact gcggtctatt actgtacaag atggggttac 360gacagggagt ggtttgctta ctggggccaa gggactctgg tcactgtctc ttcagcgtcg 420accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480gcggccctgg gctgcctggt caaggactac ttccccgaac ctgtgacggt ctcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 720tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta ccagagcacg 960taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1140aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1380agcctctccc tgtccccggg taaatga 140767717DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 67atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acatggggat 60attgtgatga ctcagtctcc actctctcta cctgtcactc ctggagagcc agcatccatc 120tcctgcaggt ctagtaagag tctcctgcat agtaatggca acacttactt gtattggttc 180ctgcagaagc caggccagtc tcctcagctc ctgatatatc ggatgtccaa ccttgcctca 240ggagtcccag acaggttcag tggcagtggg tcaggaactg ctttcacact gaaaatcagt 300agagtggagg ctgaggatgt gggtgtttat tactgtatgc aacatctaga atatccattc 360acgttcggcc aggggacaaa gctggaaata aaacgtacgg tggctgcacc atctgtcttc 420atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 480aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 540ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 600agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttag 717681456DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 68atgggctggt cctgcatcat cctgtttctg gtcgccaccg ccaccggcgt gcactcccag 60gtccagctgg tccagtctgg cgccgaagtg aagaaacctg gcgcctccgt gaaggtgtcc 120tgcaaggcct ccggctacac cttcaccagc tactacatgt actgggtccg acaggcccca 180ggccagggac tggaatggat gggcggcatc aacccctcca acggcggcac caacttcaac 240gagaagttca agtccagagt gaccatgacc accgacacct ccacctccac cgcctacatg 300gaactgcggt ccctgcggag cgacgacacc gccgtgtact actgcaccag atggggctac 360gacagagagt ggttcgccta ctggggccag ggcaccctgg tcacagtgtc ctccgcttcc 420accaagggcc cctccgtgtt ccctctggcc ccctccagca agtccacctc tggcggcacc 480gctgccctgg gctgcctggt caaagactac ttccccgagc ccgtgaccgt gtcctggaac 540tctggcgccc tgaccagcgg cgtgcacacc ttccctgccg tgctgcagtc ttccggcctg 600tactccctgt cctccgtggt caccgtgccc tccagctctc tgggcaccca gacctacatc 660tgcaacgtga accacaagcc ctccaacacc aaggtggaca agcgggtgga acccaagtcc 720tgcgacaaga cccacacctg tcccccctgc cctgcccctg aactgctggg cggaccttcc 780gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840acctgcgtgg tggtggacgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900gacggcgtgg aagtgcacaa cgccaagacc aagcccagag aggaacagta ccagtccacc 960taccgggtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaagagtac 1020aagtgcaagg tctccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080aagggccagc cccgcgagcc ccaggtgtac acactgcccc ctagccggga agagatgacc 1140aagaaccagg tgtccctgac ctgtctggtc aaaggcttct acccctccga cattgccgtg 1200gaatgggagt ccaacggcca gcccgagaac aactacaaga ccaccccccc tgtgctggac 1260tccgacggct cattcttcct gtactccaag ctgaccgtgg acaagtcccg gtggcagcag 1320ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380tccctgtccc tgagccccgg caagtgatga gtatacctgg gcctcatggg ccttcctttc 1440actgcccgct ttccag 145669832DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 69cgaattggcg gaaggccgtc aaggccacgt gtcttgtcca gagctcgata tcgccacc 58atg ggc tgg tcc tgc atc atc ctg ttt ctg gtc gcc acc gcc acc ggc 106Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac ggc gac atc gtg atg acc cag tcc ccc ctg tcc ctg ccc gtg 154Val His Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25 30 aca cct ggc gag cct gcc tcc atc tcc tgc cgg tcc tcc aag tcc ctg 202Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu 35 40 45 ctg cac tcc aac ggc aat acc tac ctg tac tgg ttc ctg cag aag ccc 250Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro 50 55 60 ggc cag tcc cct cag ctg ctg atc tac cgg atg tcc aac ctg gcc tcc 298Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser 65 70 75 80 ggc gtg ccc gac aga ttc tcc ggc tct ggc tct ggc aca gcc ttc acc 346Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr 85 90 95 ctg aag atc tcc cgg gtg gaa gcc gag gac gtg ggc gtg tac tac tgc 394Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 100 105 110 atg cag cac ctg gaa tac ccc ttc acc ttc ggc cag ggc acc aag ctg 442Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu 115 120 125 gaa atc aag cgg acc gtg gcc gct ccc tcc gtg ttc atc ttc cca ccc 490Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtc gtc tgc ctg ctg 538Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 aac aac ttc tac ccc cgc gag gcc aag gtg cag tgg aag gtg gac aac 586Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 gcc ctg cag tcc ggc aac tcc cag gaa tcc gtc acc gag cag gac tcc 634Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 aag gac agc acc tac tcc ctg tct tcc acc ctg acc ctg tcc aag gcc 682Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 gac tac gag aag cac aag gtg tac gcc tgc gaa gtg acc cac cag ggc 730Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 ctg tcc agc ccc gtg acc aag tcc ttc aac cgg ggc gag tgc 772Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 tgatgattaa ttaaggtacc tggagcacaa gactggcctc atgggccttc cgctcactgc 832

Claims

1-42. (canceled)

43. A humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: i. the VH region comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 comprises SEQ ID NO: 3, VH CDR2 comprises SEQ ID NO: 4 and VH CDR3 comprises SEQ ID NO: 5; ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein: a. the VH FWR1 comprises SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23; b. The VH FWR2 comprises SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:24 or SEQ ID NO: 50; c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and d. VH FWR4 comprises SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3 wherein the VL CDR1 comprises SEQ ID NO: 6, VH CDR2 comprises SEQ ID NO: 7 and VH CDR3 comprises SEQ ID NO: 8; iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein: a. the VL FWR1 comprises SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35; b. VL FWR2 comprises SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36; c. VL FWR3 comprises SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and d. VL FWR4 comprises SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

44. The antibody or antigen-binding fragment of claim 43, wherein: i. the VH region comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 consists of SEQ ID NO:3, VH CDR2 consists of SEQ ID NO: 4 and VH CDR3 consists of SEQ ID NO: 5; ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein: a. the VH FWR1 consists of SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23; b. The VH FWR2 consists of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:24 or SEQ ID NO: 50; c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and d. VH FWR4 consists of SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3 wherein the VL CDR1 consists of SEQ ID NO: 6, VH CDR2 comprises SEQ ID NO: 7 and VH CDR3 consists of SEQ ID NO: 8; iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein: a. the VL FWR1 consists of SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35; b. VL FWR2 consists of SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36; c. VL FWR3 consists of SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and d. VL FWR4 consists of SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38.

45. The antibody or antigen-binding fragment of claim 43, wherein the VH region comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49.

46. The antibody or antigen-binding fragment of claim 43, wherein the VL region comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

47. The antibody or antigen-binding fragment of claim 43, wherein the VH region consists of a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49.

48. The antibody or antigen-binding fragment of claim 43, wherein the VL region consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO:13 and SEQ ID NO: 14.

49. The antibody or antigen-binding fragment of claim 43, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 12.

50. The antibody or antigen-binding fragment of claim 43, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 13.

51. The antibody or antigen-binding fragment of claim 1, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 14.

52. The antibody or antigen-binding fragment of claim 43, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 54 and VL region consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

53. A humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region comprising a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49.

54. The antibody or antigen-binding fragment of claim 43, wherein the isotype of the constant region is IgG1, IgG2, IgG3, or IgG4.

55. The antibody or antigen-binding fragment of claim 54, wherein the isotype of the IgG constant region is IgG1.

56. The antibody or antigen-binding fragment of claim 54, wherein the isotype of the IgG constant region is IgG4.

57. The antibody or antigen-binding fragment of claim 43 having a heavy chain immunoglobulin constant domain selected from the group consisting of a human IgG1 constant domain and a human IgG4 constant domain.

58. The antibody or antigen-binding fragment of claim 43 having a human Ig kappa constant domain.

59. The antibody or antigen-binding fragment of claim 43, wherein the antibody is aglycosylated.

60. The antibody or antigen-binding fragment of claim 43 having a human IgG1 constant domain that is aglycosylated by replacing the amino acid corresponding to asparagine (Asn) 297 of the constant region heavy chain with an alternative amino acid residue.

61. The antibody or antigen-binding fragment of claim 60, wherein the Asn 297 is replaced with glutamine, alanine, histidine or glycine.

62. The antibody or antigen-binding fragment of claim 61, wherein the Asn 297 is replaced with glutamine.

63. The antibody or antigen-binding fragment of claim 43, wherein the heavy chain immunoglobulin constant domain is a human IgG4 constant domain wherein serine 228 is replaced with proline.

64. The antibody or antigen-binding fragment of claim 43, which is a scFv, diabody, Fab, minibody or scFv-Fc.

65. A nucleotide sequence encoding the antibody or antigen-binding fragment of claim 43.

66. A nucleotide sequence encoding a VH region, wherein the VH region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49.

67. (canceled)

68. A nucleotide sequence encoding a VL region, wherein the VL region comprises an amino acid selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

69-71. (canceled)

72. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antibody or antigen-binding fragment of claim 43.

73. A method of treating an inflammatory disease or disorder comprising administering to a subject an effective amount of the antibody or antigen-binding fragment of claim 43.

74. The method of claim 73, wherein the inflammatory disease or disorder is ischemia-reperfusion injury.

75-76. (canceled)

77. The method of claim 73, wherein the ischemia-reperfusion results after myocardial infarction, stroke or a surgical procedure.