LIBRARIES, ARRAYS AND THEIR USES FOR TARGETED AFFINITY ENHANCEMENT

Abstract

lates to methods and materials for enhancing the binding affinity of an antibody by means of generating a library or an array of targeted amino acid changes (e.g., mutations) at one or more positions in an antibody variable domain. The present disclosure relates to libraries or arrays and their uses for enhancing antibody affinity. The present disclosure relates to methods and materials for mutagenesis, including for the generation of novel or improved antibody variable domains and libraries or arrays of mutant antibody variable domains or nucleic acids encoding such mutant or modified variable domains.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of International Patent Application No. PCT/US2008/088639, filed on Dec. 31, 2008; U.S. Provisional Application No. 61/018,113, filed Dec. 31, 2007; U.S. Provisional Application No. 61/018,105, filed Dec. 31, 2007; and U.S. Provisional Application No. 61/018,101, filed Dec. 31, 2007, the disclosures of which are herein incorporated by reference in their entirety.

FIELD

[0002]The present disclosure relates to libraries or arrays and their uses for enhancing the binding affinity of an antibody. Modified antibody variable domains obtained by novel targeted affinity enhancement methods demonstrate an increased binding affinity compared to the binding affinity exhibited by the unmodified (parent) variable domain. The present disclosure also relates to novel combinations of degenerate codons that code for an equal representation of one or more non-redundant amino acid changes.

BACKGROUND

[0003]Affinity enhancement of a monoclonal antibody (beyond the ordinary nanomolar affinity which is typically achieved in an animal system) is desirable when producing a therapeutic agent, regardless of how the antibody was originally generated (e.g., by transgenic mice, by phage display, by yeast display, or by ordinary murine hybridoma methods). Extremely high affinity antibodies (e.g., a scFv or Fab) may be advantageous if they can be administered with equivalent efficacy in much lower doses, thereby decreasing the cost of producing the drug and/or diminishing its adverse side-effects.

[0004]Although natural immunological systems typically yield antibodies of nanomolar (10^-9 M) affinity, greater affinities may be desirable. However, since astronomical numbers of different antibody combining sites are possible, it has been difficult to design a method for choosing a few key mutations in an antibody variable domain which might lead to greater binding affinity, particularly in the absence of reliable structural (e.g., x-ray crystallographic) data. Present techniques for enhancing the affinity of an antibody often require screening a large number of antibody variants and may introduce undesirable mutations outside of the antibody binding pocket.

SUMMARY

[0005]The present disclosure relates to methods and materials for enhancing the binding affinity of an antibody by means of generating a library or array of targeted amino acid changes (e.g., mutations) at one or more positions in an antibody variable region to enhance affinity. For the methods, in some embodiments, antibody variable region sequences may be aligned according to a standard numbering system such as Kabat. The present disclosure relates to libraries or arrays and their uses for enhancing antibody affinity. The present disclosure also relates to novel combinations of degenerate codons which code for an equal representation of one or more non-redundant amino acid changes.

[0006]Methods are disclosed which minimize the total number of amino acid changes for enhancement of an antibody's affinity. Such methods may make a number of amino acid changes at an original amino acid position. Further, groups of positions on an antibody variable region comprising a heavy and/or light chain variable region may be selected for change by employing novel methods which assign each amino acid on the variable region of the heavy and/or light chains of antibodies to one of the following unique groups: contacting (C), peripheral (P), supporting (S), interfacial (I), or distant (D). These novel proximity groups permit the selection of amino acid residues that are candidates for change. Additionally or alternatively, positions for amino acid changes may be based upon a novel method of determining the degree to which the original amino acid residue differs from the corresponding consensus or germline residue in terms of charge, size or chemical functionality. For example, the methods provided by the disclosure may include utilization of tables of numerical components, which can be added together to identify "conspicuous" amino-acid changes.

[0007]Methods are also disclosed for enhancing the affinity of a variable region of an antibody (e.g., a heavy chain and/or light chain variable region) by identifying the proximity assigned to amino acid positions in the variable region of the antibody using the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D and preferably changing one or more contacting (C), supporting (S), peripheral (P) and/or interfacial (I) amino acid residues, with other amino acids residues. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0008]An exemplary method for affinity enhancement of an antibody variable region (e.g., a heavy chain and/or light chain variable region) includes aligning a variable region sequence with consensus or individual light-chain and heavy-chain sequences according to a standard numbering system such as Kabat; additionally or alternatively co-aligning with the antibody's own direct germline precursor sequences if they are known and preferably changing one or more contacting (C), supporting (S), peripheral (P) and/or interfacial (I) amino acid residues, with other amino acids residues. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0009]Methods are provided for enhancing the binding affinity of a variable domain (e.g., a heavy chain and/or light chain variable region) of an antibody, to obtain a modified variable domain with enhanced binding affinity by using the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D; identifying the proximity assigned to amino acid positions in the variable domain of the antibody as contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D); changing one or more contacting (C), supporting (S), peripheral (P) interfacial (I) and/or distant (D) amino acid residues, with other amino acids residues with other amino acid residues, thereby generating a library or array of modified variable domains; screening the library or array for binding affinity to a binding partner; and obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0010]In some embodiments, the other amino acid residues are alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) or valine (Val, V).

[0011]In some embodiments, the other amino acid changes can be introduced by mutagenesis (e.g., PCR-based, Dpn1-based or Kunkel mutagenesis) using primers. Exemplary primers may comprise degenerate codons, including, for example, 2 to 12-fold degenerate codons. In preferred embodiments, the degenerate codons do not encode for cysteine or methionine. In some embodiments, basic amino acid changes may be introduced using the degenerate codon ARG (R=A/G), which codes for arginine/lysine. In other embodiments, polar amino acid changes may be introduced using the degenerate codons WMC (W=A/T; M=A/C), which codes for serine/threonine/asparagine/tyrosine and/or CAS (S=C/G), which codes for histidine/glutamine. In other embodiments, acidic amino acid changes may be introduced using the degenerate codon GAS (S=C/G), which codes for glutamic acid/aspartic acid. In other embodiments, non-polar changes may be introduced using the degenerate codons NTC (N=A/G/C/T), which codes for leucine/phenylalanine/isoleucine/valine, KGG (K=G/T), which codes for tryptophan/glycine and/or SCG (S=C/G), which codes for proline/alanine. An exemplary 7 primer set includes ARG, WMC, CAS, GAS, NTC, KGG and SCG which collectively encode eighteen amino acids excluding cysteine and methionine. Alternate degenerate codons can be utilized to produce eighteen amino acids. For example, in the example of degenerate codons given above, ARG can be replaced with ARA, WMC can be replaced with WMT, CAS can be replaced with CAK (K=G,T), CAM (M=A or C), or CAW (W=A or T), NTC with NTT, SCG with SCA, SCC, or SCT. In addition, the single primer listed as NTC or NTT can be replaced with: two primers MTC, KTC (or MTT/KTT; MTC/KTT; MTT/KTC); STC, WTC (or STT/WTT; STT/WTC; STC/WTT); RTC, YTC (or RTT/YTT; RTC/YTT, RTT/YTC).

[0012]In some embodiments, degenerate codons may include, for example, NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T). An exemplary three primer set includes NHT, VAA and BGG which collectively encode eighteen amino acids excluding cysteine and methionine. Alternate degenerate codons can be utilized to produce eighteen amino acids and there are multiple codon sets that can be utilized. Either NHT or NHC (where N=A/G/C/T, H=A/C/T) can be utilized in combination with either VAG or VAA (where V=A/C/G) and either BGG or DGG (where B=C/G/T, D=A/G/T). In addition, the NHT primer can be broken up into a multitude of different degenerate primer sets. N can be broken up into B (CGT)+A; D (AGT)+C; H (ACT)+G; V (ACG)+T; K+M; S+W; R+Y; K+A+C; M+G+T; S+A+T; W+C+G; R+C+T; Y+G+A; A+C+G+T. For the second and third positions in the codon, the HT or HC would continue to be utilized. If the first codon remains either N; K+M; S+W; or R+Y, then, H can be further broken down into A+Y; C+W; T+M; or A+C+T. The third position in the codon would remain T or C.

[0013]Methods are also provided for making a modified variable domain (e.g., a heavy chain and/or light chain variable region) of an antibody with enhanced binding affinity compared to a parent variable domain by modifying the nucleotide sequence of an antibody variable domain at amino acid residues that encode preferably one or more contacting (C), peripheral (P), supporting (S), and/or interfacial (I) amino acid residues identified from the "prox" line as shown in FIGS. 3A, 3B, 3C and/or 3D to produce amino acid changes at the position, thereby generating a library of modified antibody variable domains; and selecting a modified variable domain from the library that has enhanced binding affinity. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed according to disclosed methods.

[0014]In some embodiments, the method further comprises contacting a parent variable domain with a binding partner under conditions that permit binding; contacting modified variable domain(s) with the binding partner under conditions that permit binding; and determining binding affinity of the modified variable domain(s) and the parent variable domain for the binding partner, wherein modified variable domain(s) that have a binding affinity for the binding partner greater than the binding affinity of the parent variable domain for the binding partner are identified as having enhanced binding affinity for the binding partner.

[0015]Methods are also provided for selecting a modified variable domain (e.g., a heavy chain and/or light chain variable region) of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain by obtaining a library of modified antibody variable domains comprising amino acid changes at preferably multiple (e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18) contacting (C), peripheral (P), supporting (S), and/or interfacial (I) amino acid residues identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D; determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain. For example, basic amino acid changes can be introduced (e.g., arginine (Arg, R) and/or lysine (Lys, K), polar amino acid changes can be introduced (e.g., serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), tyrosine (Tyr, Y), histidine (His, H) and/or glutamine (Gln, Q)), acidic amino acid changes can be introduced (e.g., glutamic acid (Glu, E), and/or aspartic acid (Asp, D)), and/or non-polar amino acids can be introduced (e.g., leucine (Leu, L), phenylalanine (Phe, F), isoleucine (Ile, I), valine (Val, V), tryptophan (Trp, W), glycine (Gly, G), proline (Pro, P) and/or alanine (Ala, A)).

[0016]Methods are also provided for enhancing the binding affinity of a variable domain (e.g., a heavy chain and/or light chain variable region) of an antibody, to obtain a modified variable domain with enhanced binding affinity by using the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D; identifying the proximity assigned to amino acid positions in the variable domain of the antibody as contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D); preferably changing one or more contacting (C) amino acid residues with other amino acid residues, thereby generating a library or array of modified variable domains; screening the library or array for binding affinity to a binding partner; and obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0017]Methods are provided for producing a nucleic acid library with an equal representation of one or more non-redundant amino acid changes at each of one or more positions (e.g., contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions) in a parent nucleic acid by providing a set of primers (e.g., 3, 7 or 9 primers) that each comprise at least one degenerate codon (e.g., 2 to 12-fold degenerate) at identical positions, wherein the primers are complementary to a sequence in the parent nucleic acid and the primers code for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; repeating the hybridization and amplification steps with remaining primers from the set; pooling the nucleic acids produced with each primer; and obtaining a library of nucleic acids coding for an equal representation of one or more amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0018]A set of primers is provided that comprise at least one degenerate codon at identical positions (e.g., contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions), wherein the degenerate codons code for an equal representation of one or more non-redundant amino acid changes at each of one or more positions in the parent nucleic acid and the primers are complementary to a sequence in the parent nucleic acid, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0019]A kit is also provided for mutagenesis of one or more positions in a parent nucleic acid (e.g., contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions), a set of primers comprising at least one degenerate codon at identical positions, wherein the degenerate codons code for an equal representation of one or more non-redundant amino acid changes at each of one or more positions in the parent nucleic acid and the primers are complementary to a sequence in the parent nucleic acid, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0020]In some embodiments, the primer set codes for eighteen amino acid changes at each of one or more positions in the parent nucleic acid. In some embodiments, the set of primers each comprise a degenerate codon which collectively code for alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine at each position. In some embodiments, the set of primers comprises three primers. In some embodiments, the primers each comprise one or more degenerate codons as represented by NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T). In some embodiments, the set of primers comprises seven primers. In some embodiments, the primers each comprise one or more degenerate codons as represented by ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

[0021]In some embodiments, the primer set codes for basic amino acid changes at each of one or more positions in the parent nucleic acid. In some embodiments, the primer set comprises one primer. In some embodiment, the one primer comprises a degenerate codon which codes for arginine and lysine. In some embodiments, the one primer comprises one or more degenerate codons as represented by ARG (where, R=A/G).

[0022]In some embodiments, the primer set codes for polar amino acid changes at each of one or more positions in the parent nucleic acid. In some embodiments, the primer set comprises two primers. In some embodiments, the two primers each comprise a degenerate codon which collectively code for serine, threonine, asparagine and tyrosine. In some embodiments, the two primers each comprise one or more degenerate codons as represented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

[0023]In some embodiments, the primer set codes for acidic amino acid changes at each of one or more positions in the parent nucleic acid. In some embodiments, the primer set comprises one degenerate codon. In some embodiments, the one primer comprises a degenerate codon that codes for glutamic acid and aspartic acid. In some embodiments, the one primer comprises one or more degenerate codons as represented by GAS (where S=C/G).

[0024]In some embodiments, the primers code for non-polar amino acid changes at each of one or more positions in the parent nucleic acid. In some embodiments, the primer set comprises three degenerate codons. In some embodiments, the three primers each comprise a degenerate codon that collectively code for glutamic acid and aspartic acid. In some embodiments, the primers each comprise one or more degenerate codons as represented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G).

[0025]In some embodiments, the parent nucleic acid encodes an antibody variable region. In some embodiments, the positions in the parent nucleic acid code for contacting (C), supporting (S), interfacial (I), peripheral (P) or distant (D) residues.

[0026]In some embodiments, the contacting (C) residue may be in complementarity determining domain-1 (CDR1) in a light chain variable domain. In certain embodiments, the contacting (C) residue may be at a position corresponding to position 28, 30 or 31 in CDR1. In other embodiments, the contacting (C) residue may be in CDR2 in a light chain variable domain. In certain embodiments, the contacting (C) residue may be at a position corresponding to position 50, 51 or 53 in CDR2. In other embodiments, the contacting (C) may be in CDR3 in a light chain variable region. In some embodiments, the contacting (C) residue may be in CDR1 in a heavy chain variable domain. In certain embodiments, the contacting (C) residue may be at a position corresponding to position 32 or 33 in CDR1. In some embodiments, the (C) contacting residue may be in CDR2 in a heavy chain variable domain. In certain embodiments, the contacting (C) residue may be at a position corresponding to position 50, 52, 53, 54, 56, or 58 in CDR2. In some embodiments, the contacting (C) may be in CDR3 in a heavy chain variable region.

[0027]In some embodiments, the methods further comprise inserting the modified antibody variable domain into an appropriate vector. In some embodiments, the vector is a plasmid, phage or phagemid. In certain embodiments, the vector is pXOMA Fab or pXOMA-gIII-Fab (see, e.g., FIG. 6). The pXOMA Fab vector is similar to the pXOMA-gIII-Fab vector but does not have a pIII coding sequence.

[0028]In some embodiments, the variable domain is from a chimeric antibody. In other embodiments, the variable domain is from a humanized or human engineered antibody. In some embodiments, the variable domain is from a human antibody.

[0029]In some embodiments, binding affinity of a modified variable domain or parent variable domain to a binding partner is determined by measuring K_off. In some embodiments, binding affinity of a modified variable domain or parent variable domain to a binding partner may be measured by Biacore (e.g., Biacore 2000 or A100).

[0030]The present disclosure also provides method of mutagenesis of a parent nucleic acid encoding an antibody variable domain to generate modified antibody variable domains by obtaining one or more primers that each comprise at least one 2 to 12 fold degenerate codon, wherein each primer comprises at least two oligonucleotide sequences that are complementary to a sequence in the parent nucleic acid and code for an amino acid mutation with the exception of cysteine or methionine at one amino acid position encoded by the parent nucleic acid; and mutating the parent nucleic acid by replication or polymerase based amplification using the one or more obtained primers, wherein replication or amplification of the parent nucleic acid with the one or more primers generates mutated nucleic acids that encode modified antibody variable domains.

[0031]The present disclosure also provides methods for mutagenesis of an antibody variable domain to obtain modified antibody variable domains with mutated amino acid sequences by identifying one or more amino acid positions in the antibody variable domain for mutagenesis; substituting one or more of the identified amino acid residues in the antibody variable domain with other amino acid residues excluding cysteine and methionine to generate a library or an array of modified antibody variable domains with mutated amino acid sequences; screening the library or array of modified antibody variable domains in an assay for a biological activity of the antibody variable domain; and obtaining modified antibody variable domains having the biological activity of the antibody variable domain.

[0032]The present disclosure also provides for generating an array of nucleic acids encoding modified antibody variable domains by obtaining a collection of nucleic acids encoding modified antibody variable domains containing amino acid mutations other than cysteine and methionine at amino acid residues of a parent antibody variable domain sequence by mutagenesis of a nucleic acid encoding the antibody variable domain sequence using primers that each comprise at least one 2 to 12 fold degenerate codon; sequencing the collection of nucleic acids encoding the modified antibody variable domains; and arranging each sequenced nucleic acid encoding a modified antibody variable domain to generate an array of nucleic acid sequences each encoding a modified antibody variable domain.

[0033]The present disclosure also provides methods for generating an array of nucleic acid sequences encoding modified antibody variable domains by preparing a plurality of nucleic acid sequences by mutagenesis that encode a plurality of modified antibody variable domains that vary from a parent antibody variable domain sequence at one or more amino acid positions and contain one of eighteen different amino acids excluding cysteine and methionine at each position mutated from the parent protein sequence; and arranging each nucleic acid sequence prepared in step (a) to generate an array of nucleic acid sequences each encoding a modified antibody variable domain.

[0034]The present disclosure also provides methods for generating an array of clones comprising nucleic acids encoding modified antibody variable domains by preparing a plurality of nucleic acids by mutagenesis that encode a plurality of modified antibody variable domains that vary from a parent antibody variable domain sequence at one or more amino acid positions and contain one of eighteen different amino acids excluding cysteine and methionine at each position varied from the parent antibody variable domain sequence; transfecting the prepared nucleic acids into host cells and selecting clones comprising the transfected nucleic acids; and arranging each selected clone to generate an array of clones with each arrayed clone capable of expressing a modified antibody variable domain.

[0035]The present disclosure also provides methods of producing a nucleic acid library with an equal representation of non-redundant amino acid changes at an amino acid position encoded by a parent nucleic acid encoding an antibody variable domain by providing a set of primers that each comprise at least one degenerate codon, wherein each primer comprises at least two oligonucleotide sequence that are complementary to a sequence in the parent nucleic acid and code for an amino acid mutation with the exception of cysteine and methionine at one amino acid position encoded by the parent nucleic acid, wherein the primers code for an equal representation of non-redundant amino acid changes at the one position; hybridizing a primer from the set to the parent nucleic acid; replicating or amplifying the parent nucleic acid molecule with the primer to generate nucleic acids that code for amino acid changes at the one position, repeating the hybridizing and replicating steps with each remaining primer from the set; pooling the nucleic acids produced with each primer; and obtaining a library of nucleic acids coding for an equal representation of amino acid changes at the one position.

[0036]The present disclosure also provides methods for obtaining a nucleic acid sequence with an improvement in comparison to a parent nucleic acid sequence encoding an antibody variable domain with respect to at least one molecular or biological property of interest, said method comprising; obtaining a set of primers that each comprise at least one 2 to 12 fold degenerate codon that does not code for cysteine and methionine, wherein the primers are complementary to a sequence in the parent nucleic acid sequence and wherein the primers code for non-redundant amino acid mutations at one amino acid position encoded by the parent nucleic acid sequence; mutating the parent nucleic acid sequence by replication or polymerase based amplification using the obtained set of primers to generate variant nucleic acid sequences; producing a library or array of variant nucleic acid sequences coding for amino acid mutations at the one position in the parent nucleic acid sequence; and screening the library or array of variant nucleic acid sequences to identify nucleic acid sequences that have a desirable improvement in comparison with the parent nucleic acid sequence with respect to at least one molecular or biological property of interest.

[0037]The present disclosure also provides methods of making modified antibody variable domains with mutated amino acid sequences by modifying the amino acid sequence of an antibody variable domain to produce amino acid mutations at an amino acid residue in the antibody variable domain to generate a library or an array of modified antibody variable domains with mutated amino acid sequences, wherein the amino acid mutations exclude cysteine and methionine; and selecting modified antibody variable domains from the library or the array that have a biological activity of an unmodified antibody variable domain.

[0038]The present disclosure also provides methods for selecting modified antibody variable domains with mutated amino acid sequences by obtaining a library or an array of modified antibody variable domains comprising amino acid mutations at one amino acid residues in an amino acid sequence of a protein, wherein the amino acid mutations exclude cysteine and methionine; assaying the modified antibody variable domains for a biological activity of an unmodified protein; and selecting the modified antibody variable domains that have a biological activity of the unmodified antibody variable domain.

[0039]In some embodiments, the amino acid mutations are selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine at each position.

[0040]In some embodiments, the set of primers or primers code for eighteen amino acid mutations at the one amino acid position encoded by the parent nucleic acid.

[0041]In some embodiments, three primers that each comprise at least one 2 to 12 fold degenerate codon are obtained or used. In some embodiments, seven primers that each comprise at least one 2 to 12 fold degenerate codon are obtained or used. In some embodiments, the degenerate codons are selected from the group consisting of: NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T). In some embodiments, the degenerate codons are selected from the group consisting of: ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

[0042]In some embodiments, the primers code for basic amino acid mutations at the one amino acid position encoded by the parent nucleic acid. In some embodiments, one primer that comprises at least one 2 to 12 fold degenerate codon is obtained. In some embodiments, the one primer comprises a degenerate codon which codes for arginine and lysine. In some embodiments, the degenerate codon is represented by ARG (where, R=A/G).

[0043]In some embodiments, the primers code for polar amino acid mutations at the one amino acid position encoded by the parent nucleic acid. In some embodiments, two primers that comprise at least one 2 to 12 fold degenerate codon is obtained. In some embodiments, the two primers comprise degenerate codons that collectively code for serine, threonine, asparagine and tyrosine. In some embodiments, the degenerate codons are represented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

[0044]In some embodiments, the primers code for acidic amino acid mutations at the one amino acid position encoded by the parent nucleic acid. In some embodiments, one primer that comprises at least one 2 to 12 fold degenerate codon is obtained. In some embodiments, the one primer comprises a degenerate codon that codes for glutamic acid and aspartic acid. In some embodiments, the degenerate codon is represented by GAS (where S=C/G).

[0045]In some embodiments, the primers code for non-polar amino acid mutations at the one amino acid position encoded by the parent nucleic acid. In some embodiments, three primers that comprise at least one 2 to 12 fold degenerate codon are obtained. In some embodiments, the three primers comprise degenerate codons that collectively code for glutamic acid and aspartic acid. In some embodiments, the degenerate codons are represented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G).

[0046]In some embodiments, the methods may further comprising selecting the one or more positions in the parent nucleic acid sequence for mutation.

[0047]In some embodiments, the position for mutation encodes one or more CDR residues. In some embodiments, the CDRs are defined by Kabat, Chothia or IMGT. In some embodiments, all CDR resides are mutated.

[0048]In some embodiments, modified antibody variable domains are selected that have increased activity as compared to the unmodified antibody variable domain. In some embodiments, modified antibody variable domains are selected that have decreased activity as compared to the unmodified antibody variable domain. In some embodiments, modified antibody variable domains are selected that have equal activity as compared to the unmodified antibody variable domain.

[0049]In some embodiments, the mutagenesis or substitution is performed with one or more primers that each comprise at least one 2 to 12 fold degenerate codon, wherein each primer comprises at least two oligonucleotide sequences that are complementary to a sequence in a parent nucleic acid and code for an amino acid substitution with the exception of cysteine and methionine at one amino acid position encoded by the parent nucleic acid.

[0050]The present disclosure also provides a library or an array comprising variants of a antibody variable domain sequence, wherein the variants each comprise an amino acid mutation at one amino acid position in the sequence of a parent antibody variable domain and wherein the amino acid mutations are not cysteine or methionine.

[0051]The present disclosure also provides methods for obtaining a nucleic acid sequence with an improvement in comparison to a parent nucleic acid sequence encoding an antibody variable domain with respect to at least one molecular or biological property of interest by mutating the parent nucleic acid by polymerase based amplification using one or more primers that each comprise at least one 2 to 12 fold degenerate codon to generate mutated nucleic acid sequences, wherein each primer comprises at least two oligonucleotide sequences that are complementary to a sequence in the parent nucleic acid and code for an amino acid mutation with the exception of cysteine or methionine at one amino acid position encoded by the parent nucleic acid; sequencing the mutated nucleic acid sequences; arranging each sequenced mutated nucleic acid sequence comprising one amino acid mutation to generate an array of mutated nucleic acid sequences; and screening the array of variant nucleic acid sequences to identify nucleic acid sequences that have a desirable improvement in comparison with the parent nucleic acid sequence with respect to at least one molecular or biological property of interest.

[0052]In some embodiments, modified antibody variable domains are selected that have increased activity as compared to the unmodified protein. In some embodiments, modified antibody variable domains are selected that have decreased activity as compared to the unmodified protein. In some embodiments, modified antibody variable domains are selected that have equal activity as compared to the unmodified protein.

[0053]The present disclosure also provides antibodies or binding fragments thereof made by the methods of the present disclosure.

[0054]Additional features and advantages are described herein, and will be apparent from, the following Detailed Description and the Figures.

BRIEF DESCRIPTION OF THE FIGURES

[0055]FIG. 1 is a generalized schematic map of an exemplary antibody combining site as described herein, looking downward onto the "top" surface of a variable domain comprising a light chain variable region and a heavy chain variable region. It shows the six CDR loops (L1, L2, L3, H1, H2, H3) which are spatially located directly above the three-dimensional structure of the evolutionarily-conserved framework underneath. As shown and discussed herein, this map provides roughly approximate higher-order structural information, which is not available from the linear primary sequence alone, such as the identity of potential nearest neighbors in the space-filling model of a generic variable domain. Specific features of the murine ING1 monoclonal antibody have been added to this map, so that it can also call attention to localized domains of the antibody's combining site containing clusters of high-conspicuousness positions as described herein, which are likely to be in contact with sidechains on the antigen. In particular, each amino-acid position in the murine ING1 antibody is represented on this map by a white rectangle containing a group of symbols. The letter and number at the bottom-left of each rectangle (e.g., "H 98" in CDR-loop H3) is the Kabat-position number of the amino-acid residue on the antibody molecule within either chain (L=light, H=heavy). The small upper-case letter (e.g., "B") at the bottom-right is a designation for the residue's proximity as described herein (C=Contacting, P=Peripheral, S=Supporting, I=Interfacial) relative to the antibody's binding site (shown on the "prox" line in FIGS. 2A-2D). The large upper-case letter (e.g., "A") at the upper-left is the amino-acid code for the residue's sidechain (line "murING1" in FIGS. 2A-2D). The large single digit at the upper right (e.g., "3") in some rectangles is the non-zero conspicuousness-value as described herein of affinity enhancement for the sidechain (line "cspc" in FIGS. 2A-2D), calculated in reference to the appropriate human consensus sequence for light chain (hK2) or heavy chain (hH 1). Rectangles with no such value reflect a conspicuousness of zero.

[0056]FIGS. 2A-2D: Alignments of sequences in the light chain and heavy chain, with lines (e.g., prox, cspc) relating to affinity enhancement and lines relating to human engineering (e.g., risk) are shown. In each set of lines, the top ones apply the present disclosure to the murine ING1 antibody (2A-2B), and the bottom ones relate the present disclosure to the general principles of human engineering (Studnicka et al., Protein Engineering, 7(6):805-814 (1994); U.S. Pat. No. 5,766,886). Each set of lines shows the Kabat position numbers (pos), the general classification of proximity groups for each position of every antibody (prox), the murine ING1 monoclonal antibody sequence to be affinity-enhanced (murING1), the conspicuousness value as described herein of each position for affinity-enhancement when the murine ING1 antibody is compared to murine consensus sequences (cspc), several murine consensus sequences to which ING1 is compared (mK2 or mH2a), the human ING1 residues which are introduced during the HUMAN ENGINEERING® process (humING1), the degree of disconnection of the sidechain from the antibody's combining site (disc) as described herein, the degree of outward-orientation of the sidechain on the antibody's surface (outw) as described herein, the degree of risk for human engineering (risk), and the Kabat position numbers (pos) (2A-2B). Similarly, FIGS. 2C and 2D are alignments of sequences in the light chain and heavy chain of IL-1 antibody (also referred to as cA5 and/or XPA23), with lines (e.g., prox, cspc) relating to affinity enhancement and lines relating to human engineering (e.g., risk).

[0057]FIGS. 3A-3D are mutual alignments of consensus sequences (Kabat et al. (1991) (eds), Sequences of Proteins of Immunological Interest, 5th ed.) for major murine and human subgroups of the light chain and heavy chain. Each alignment relates them to the proximity groups as described herein for each position (prox), and the Kabat position numbers (pos).

[0058]FIG. 4 shows a chart of the numerical components which can be added together to calculate each amino acid's affinity-enhancement conspicuousness value, including the components for changes in class-and-charge, for changes in physical size due to somatic mutation, and for repeated identical mutations at the same position in multiple homologous antibodies.

[0059]FIG. 5 shows PCR mutagenesis of CDR3 utilizing the CDR-H3 oligonucleotide H3-3NP2 (SEQ ID NO: 267): 5'-GCTACATATTTCTGTGCAAGATTTG GCTCTKGGGTGGACTACTGGGGTCAAGG-3', which introduces an amino acid substitution into CDR3, and the reverse primer Notl-R (SEQ ID NO: 285): 5'-AGCGGCCGCACAAGATTTGGGCTCAACTCTC-3', which incorporates the Notl restriction site into the PCR product.

[0060]FIG. 6 depicts the plasmid map of the pXOMA-gIII-Fab vector. The vector is 5,202 base pairs in length and has Ascl and Notl restriction sites flanking the heavy chain encoding sequences, and HindIII and Ascl restriction sites flanking the light chain encoding sequences. The heavy chain encoding sequences are fused to pIII encoding sequences in the vector. The pXOMA-Fab vector is similar but lacks the pIII encoding sequences.

[0061]FIG. 7 depicts the strategy for creating the light chain combination variants.

[0062]FIG. 8 depicts the strategy for creating the heavy chain combination variants.

[0063]FIG. 9A-9B shows CDR1, CDR2 and CDR3 as identified by the Kabat, Chothia and IMGT numbering scheme for ING-1 (9A) and XPA23 (9B).

[0064]FIG. 10A-10D depict a continuous numbering scheme for the heavy and light chain of XPA23 (10A and 10B, respectively). Consecutive numbering from position 1 in the light chain continues in the heavy chain such that position 1 in the heavy chain is also assigned number 108 since the light chain sequence ends at number 107. Boxed residues indicate CDRs identified by the IMGT method. FIGS. 10C and 10D show a continuous numbering scheme for the heavy and light chain of ING-1 (10C and 10D, respectively).

[0065]FIG. 11: Periplasmic extracts of clones containing one of the eighteen preferred amino acid mutations at Heavy Chain contacting positions in ING-1 were tested on Biacore for improved off-rate (see example 7). Clones with greater than 1.9-fold decrease in off-rate are listed.

[0066]FIG. 12: Periplasmic extracts of clones containing one of the eighteen preferred amino acid mutations at Light Chain contacting positions in ING-1 were tested on Biacore for improved off-rate (see example 7). Clones with greater than 1.9-fold decrease in off-rate are listed.

[0067]FIG. 13: Periplasmic extracts of clones containing one of the eighteen preferred amino acid mutations at Heavy Chain contacting positions in XPA23 were tested on Biacore for improved off-rate (see example 7). Clones with greater than 1.9-fold decrease in off-rate are listed.

[0068]FIG. 14: Periplasmic extracts of clones containing one of the eighteen preferred amino acid mutations at Light Chain contacting positions in XPA23 were tested on Biacore for improved off-rate (see example 7). Clones with greater than 1.9-fold decrease in off-rate are listed.

[0069]FIG. 15A-15D depicts two modified IgGs with an A102F or 102G substitution that were prepared and evaluated by Biacore with improved affinity (15B-15C, respectively) as compared to the parental (15A) ING-1 antibody. 15D shows the affinity determination kinetics for both the modified and parental ING-1 antibodies.

[0070]FIG. 16A-16C are sensogram profiles depicting ING-1 light chain binding to Ep-Cam.

[0071]FIG. 17 depicts modified ING-1 antibodies each comprising two or more heavy chain mutations as compared to the parental antibody. Combinations of heavy chain mutations yield affinity improvements up to 25-fold over the parental ING-1 antibody. Affinity improvements are driven largely by improvements in k_off.

[0072]FIG. 18 shows amino acid substitutions at position 32 in the light chain variable region of XPA23. Generally the substitutions at position 30 decreased kd of the antibody-antigen interaction compared to the parental antibody.

[0073]FIG. 19 shows amino acid substitutions at position 30 in the light chain variable region of XPA23. Generally the substitutions at position 30 resulted in a comparable kd of the antibody-antigen interaction compared to the parental antibody.

[0074]FIG. 20 shows amino acid substitutions at position 45 in the heavy chain variable region of XPA23. Generally the substitutions improved kd of the antibody-antigen interaction at this position compared to the parental antibody.

DETAILED DESCRIPTION

[0075]The present disclosure provides methods for enhancing the binding affinity of an antibody by means of generating a library or array of targeted amino acid changes (e.g., mutations) at one or more positions in an antibody variable domain. These methods for targeted affinity enhancement may be utilized even in the complete absence of any detailed information about the interaction between the antibody and its binding partner. The methods of the present disclosure do not require any three-dimensional x-ray crystallographic structures of the chosen antibody's combining site with its binding partner and/or any type of energy-minimization algorithm. Such targeted amino acid changes at one or more positions in an antibody variable domain that result in enhanced binding as compared to a parent variable domain may be combined in a single antibody variable domain. As used herein, array refers to an ordered arrangement of members, including, for example, clones, periplasmic extracts, cell lysates, polynucleotides or nucleic acids and polypeptides or proteins.

[0076]The present disclosure also provides methods for enhancing the affinity of an variable region of an antibody by identifying the proximity assigned to one or more amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D and preferably changing one or more contacting (C), supporting (S), peripheral (P) and/or interfacial (I) amino acid residues, with other amino acids residues. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0077]In an exemplary method to accomplish targeted affinity enhancement, amino acid residues may be selected for change by aligning a light chain or heavy chain variable region sequence of an antibody and comparing the sequence with any other variable region sequence (e.g., a homologous consensus sequence for the light and heavy chain subgroups to which it is most similar, and/or with its own precursor germline sequence if it is available). Using the sequence alignment and the "prox" line shown in FIG. 3A, 3B, 3C and/or 3D to identify the proximity assigned to amino acid positions in the variable region of a light chain and/or heavy chain as contacting (C), peripheral (P), supporting (S), interfacial (I) and/or distant (D), amino acid residues may be selected for change.

[0078]Additionally or alternatively, the primary amino-acid sequence may be characterized to identify amino acid residues that are "conspicuous" (e.g., by calculations as described herein) and that may be candidates for change. Residues differing markedly in charge or size or chemical functionality from the corresponding residues in the selected sequence, including, for example, the consensus or the germline, may confer specific affinity for antigen upon the antibody.

[0079]Amino acid positions identified as preferably contacting (C), peripheral (P), supporting (S) and/or interfacial (I) may be changed to other amino acid residues to create a library or array of modified antibody variable domains. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed. Selected amino acid residues may be changed with other naturally occurring and/or synthetic amino acid residues to create a library or multiple libraries and/or an array or multiple arrays of modified variable domains.

[0080]Modified variable domains may have one or more amino acid changes at preferably one or more contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino acid residues identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D which provides for enhanced binding affinity. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed. The library or array of modified antibody variable domains may be screened to identify those modified antibody variable domains that bind to a binding partner with increased affinity as compared to the unmodified (parent) variable domain.

[0081]The present disclosure also provides methods for producing a nucleic acid library or array with an equal representation of one or more non-redundant amino acid changes at each of one or more positions in a parent nucleic acid. Such methods may be used to introduce classes (e.g., polar, non-polar, basic and acidic) of amino acid changes at one or more positions in a parent nucleic acid. The methods may be used to introduce eighteen amino acid changes (e.g., alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) at one or more positions in a parent nucleic acid by using a set of primers comprising between three and nine primers each with a degenerate codon at an identical position. Certain amino acids may be excluded from the primer set (e.g., cysteine and methionine). Further, a set of non-redundant degenerate codons (e.g., collectively coding for eighteen codons) permits an over-representation of substitutions at each position compared to libraries of the same size created using redundant degenerate codons (e.g., degenerate codons that individually or collectively code for thirty-two or sixty-four codons). This over-representation of amino acid substitutions results in a smaller library size with greater diversity. Without being bound by a theory of the invention, it is hypothesized that the use of the above degenerate codons can allow evaluation of how side chain functionalities affect the binding interaction with the target at the positions of interest (e.g., contacting positions, etc.). For example, the use of the ARG codon can probe the effect of a positive charge upon the affinity of the antibody towards the target. Similarly the GAS codon can probe the effect of a negative charge, the WMC and CAS codons a polar substitution, and the NTC, KGG and SCG codons a non-polar substitution.

[0082]Methods for producing a nucleic acid library or array with an equal representation of eighteen non-redundant amino acid changes at each of one or more contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions in a parent nucleic acid encoding an antibody variable domain may comprise providing a set of three primers that each comprise one or more degenerate codons as represented by NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T), wherein the primers are complementary to a sequence in the parent nucleic acid and the primers code for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; repeating the hybridization and amplification steps with remaining primers from the set; pooling the nucleic acids produced with each primer; and obtaining a library or array of nucleic acids coding for an equal representation of eighteen amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0083]Methods for producing a nucleic acid library or array with an equal representation of eighteen non-redundant amino acid changes at each of one or more contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions in a parent nucleic acid encoding an antibody variable domain may comprise providing a set of seven primers that each comprise one or more degenerate codons as represented by ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G), wherein the primers are complementary to a sequence in the parent nucleic acid and the primers code for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; repeating the hybridization and amplification steps with remaining primers from the set; pooling the nucleic acids produced with each primer; and obtaining a library or array of nucleic acids coding for an equal representation of eighteen amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0084]Methods for producing a nucleic acid library or array with an equal representation of non-redundant basic amino acid changes at each of one or more contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions in a parent nucleic acid encoding an antibody variable domain may comprise providing a set of one primer that comprises one or more degenerate codons as represented by ARG (where, R=A/G), wherein the primer is complementary to a sequence in the parent nucleic acid and the primer codes for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; and obtaining a library or array of nucleic acids coding for an equal representation of basic amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0085]Methods for producing a nucleic acid library or array with an equal representation of non-redundant acidic amino acid changes at each of one or more contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions in a parent nucleic acid encoding an antibody variable domain may comprise providing a set of one primer that comprises one or more degenerate codons as represented by GAS (where S=C/G), wherein the primer is complementary to a sequence in the parent nucleic acid and the primer codes for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; and obtaining a library or array of nucleic acids coding for an equal representation of acidic amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0086]Methods for producing a nucleic acid library or array with an equal representation of non-redundant polar amino acid changes at each of one or more contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) positions in a parent nucleic acid encoding an antibody variable domain may comprise providing a set of two primers that each comprise one or more degenerate codons as represented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G), wherein the primers are complementary to a sequence in the parent nucleic acid and the primers code for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; repeating the hybridization and amplification steps with remaining primers from the set; pooling the nucleic acids produced with each primer; and obtaining a library or array of nucleic acids coding for an equal representation of polar amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0087]Methods for producing a nucleic acid library or array with an equal representation of non-redundant non-polar amino acid changes at each of one or contacting (C), peripheral (P), supporting (S), interfacial (I) or distant (D) more positions in a parent nucleic acid encoding an antibody variable domain may comprise providing a set of three primers that each comprise one or more degenerate codons as represented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G), wherein the primers are complementary to a sequence in the parent nucleic acid and the primers code for an equal representation of non-redundant amino acid changes at one or more positions; hybridizing a primer from the set to the parent nucleic acid; amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; repeating the hybridization and amplification steps with remaining primers from the set; pooling the nucleic acids produced with each primer; and obtaining a library or array of nucleic acids coding for an equal representation of non-polar amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0088]The present disclosure also provides an ING-1 heavy chain variable region as set forth in SEQ ID NO: 579 that comprises a substitution at position 28 or 30 in HCDR1. In some embodiments, the substitution at position 28 is selected from the group consisting of: T28V, T281 and T28P. In some embodiments, the substitution at position 30 is T30Y.

[0089]The present disclosure also provides an ING-1 heavy chain variable region as set forth in SEQ ID NO: 579 that comprises a substitution at position 59 in HCDR2. In some embodiments, the substitution at position 59 is T59W.

[0090]The present disclosure also provides an ING-1 heavy chain variable region as set forth in SEQ ID NO: 579 that comprises a substitution at position 100, 101 or 102 in HCDR3. In some embodiments, the substitution at position 100 is G100R. In some embodiments, the substitution at position 101 is selected from the group consisting of: S101K, S101Q, S101V, S101I, S101G. In some embodiments, the substitution at position 102 in HCDR3 is selected from the group consisting of: A102R, A102H, A102Y, A102W, A102F and A102G.

[0091]The present disclosure also provides an ING-1 light chain variable region as set forth in SEQ ID NO: 580 that comprises a substitution at position 28 or 29 in LCDR1. In some embodiments, the substitution at position 28 in LCDR1 is selected from the group consisting of: S28R, S28K, S28H, S28Y, S28F, S28Q, S28V, S28I and S28L. In some embodiments, the substitution at position 29 in LCDR1 is selected from the group consisting of L29S and L29A.

[0092]The present disclosure also provides an ING-1 light chain variable region as set forth in SEQ ID NO: 580 that comprises a substitution at 54, 55 or 58 in LCDR2. In some embodiments, the substitution at position 54 in LCDR2 is selected from the group consisting of: Y54K and Y54L. In some embodiments, the substitution at position 55 in LCDR2 is selected from the group consisting of: Q55R, Q55H and Q55W. In some embodiments, the substitution at position 58 in LCDR2 is selected from the group consisting of: N58W, N58V, N58I and N58P.

[0093]The present disclosure also provides an ING-1 light chain variable region as set forth in SEQ ID NO: 580 that comprises a substitution at position 97, 98, 99 or 100 in LCDR3. In some embodiments, the substitution at position 97 in LCDR3 is L97I. In some embodiments, the substitution at position 98 in LCDR3 is selected from the group consisting of: E98R, E98K, E98T, E98S and E98L. In some embodiments, the substitution at position 99 in LCDR3 is L99I. In some embodiments, the substitution at position 100 in LCDR3 is P100Y.

[0094]The present disclosure also provides an ING-1 antibody that comprises a heavy chain variable region as set forth in SEQ ID NO: 579 and a light chain variable region as set forth in SEQ ID NO: 580, wherein the heavy chain variable region and/or light chain variable region comprise one or more of the substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3 as disclosed herein.

[0095]The present disclosure also provides an XPA-23 light chain variable region as set forth in SEQ ID NO: 582 that comprises a substitution at position 27, 28 or 29 in LCDR1. In some embodiments, substitution at position 27 in LCDR1 is selected from the group consisting of: Q27S, Q27F and Q27G. In some embodiments, the substitution at position 28 in LCDR1 is selected from the group consisting of: D281, D28S and D28W. In some embodiments, the substitution at position 30 in LCDR1 is N30F.

[0096]The present disclosure also provides an XPA-23 light chain variable region as set forth in SEQ ID NO: 582 that comprises a substitution at position 51 or 53 in LCDR2. In some embodiments, the substitution at position 51 in LCDR2 is A51G. In some embodiments, the substitution at position 53 in LCDR2 is selected from the group consisting of: S53K and S53R.

[0097]The present disclosure also provides an XPA-23 light chain variable region as set forth in SEQ ID NO: 581 that comprises a substitution at position 92, 93, 95 or 96 in LCDR3. In some embodiments, the substitution at position 92 in LCDR3 is D92S. In some embodiments, the substitution at position 93 in LCDR3 is selected from the group consisting of: S93D and S93E. In some embodiments, the substitution at position 95 in LCDR3 is selected from the group consisting of: P95S and P95A. In some embodiments, the substitution at position 96 in LCDR3 is L96W.

[0098]The present disclosure also provides an XPA-23 heavy chain variable region as set forth in SEQ ID NO: 581 that comprises a substitution at position 135, 138, 139, 140 or 142 in HCDR1. In some embodiments, the substitution at position 135 in HCDR1 is selected from the group consisting of: T135K and T135E. In some embodiments, the substitution at position 138 in HCDR1 is selected from the group consisting of: K138Y, K138W, K138E, K138L, K138P and K138H. In some embodiments, the substitution at position 139 in HCDR1 is Y139H. In some embodiments, the substitution at position 140 in HCDR1 is F1401. In some embodiments, the substitution at position 142 in HCDR1 is selected from the group consisting of: F142T and F142A.

[0099]The present disclosure also provides an XPA-23 heavy chain variable region as set forth in SEQ ID NO: 581 that comprises a substitution at position 161 or 163 in HCDR2. In some embodiments, the substitution at position 161 in HCDR2 is selected from the group consisting of: S161R and S161K. In some embodiments, the substitution at position 163 in HCDR2 is selected from the group consisting of: G163L, G163Q, G163W, G163Y, G163I, G163K, G163R and G163F.

[0100]The present disclosure also provides an XPA-23 heavy chain variable region as set forth in SEQ ID NO: 581 that comprises a substitution at position 208, 210, 211 or 212 in HCDR3. In some embodiments, the substitution at position 208 in HCDR3 is Y208L. In some embodiments, the substitution at position 210 in HCDR3 is G210V. In some embodiments, the substitution at position 211 in HCDR3 is selected from the group consisting of: N211A and N211V. In some embodiments, the substitution at position 212 in HCDR3 is selected from the group consisting of: S212E and S212P.

[0101]The present disclosure also provides an XPA-23 antibody that comprises a heavy chain variable region as set forth in SEQ ID NO: 581 and a light chain variable region as set forth in SEQ ID NO: 582, wherein the heavy chain variable region and/or light chain variable region comprise one or more of the substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3 as disclosed herein.

[0102]Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are described.

Characterization of Amino Acid Residues in an Antibody Variable Domain

[0103]The present disclosure provides novel methods to assign each amino acid in an antibody heavy and/or light chain variable region to one of the following unique groups, which includes, contacting (C), peripheral (P), supporting (S), interfacial (I), or distant (D) residues, as shown, for example, on the "prox" lines of FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D. For example, each of the more-than-200 amino-acid positions in an antibody's variable light chain and heavy chain has been designated as a member of one of these five novel groups. The "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D is useful for any variable region sequence, irrespective of the specific amino acid sequence, such that residues can be selected as candidates for change (e.g., any and/or all contacting (C) residues). Additionally or alternatively, methods are provided that identify the presence of conspicuous amino-acid residues which may be candidates for change. Conspicuous amino acid changes may differ in charge or size or chemical functionality from the corresponding residues in the selected sequence (e.g., consensus or germline sequence) and represent positions where amino acid changes may enhance affinity.

[0104]Complementarity determining regions (CDRs) in an antibody variable domain (e.g., LCDR1, LCDR2 and LCDR3 for the light chain and HCDR1, HCDR2 and HCDR3 for the heavy chain) may be defined according to any known method in the art including, for example, Kabat, Chothia or IMGT. Kabat, Chothia and IMGT definitions of CDRs 1-3 in the light chain and the heavy chain of ING-1 and XPA23 is shown in FIGS. 9A and 9B, respectively. According to Kabat, LCDR1 comprises amino acid residues 24 to 34, LCDR2 comprises amino acid residues 50 to 56, LCDR3 comprises amino acid residues 89 to 97, HCDR1 comprises amino acid residues 31 to 35b, HCDR2 comprises amino acid residues 50 to 65 and HCDR3 comprises amino acid residues 95 to 102. According to Chothia, LCDR1 comprises amino acid residues 24 to 34, LCDR2 comprises amino acid residues 50 to 56, LCDR3 comprises amino acid residues 89 to 97, HCDR1 comprises amino acid residues 26 to 32, HCDR2 comprises amino acid residues 52 to 56 and HCDR3 comprises amino acid residues 95 to 102. According to IMGT, LCDR1 comprises amino acid residues 27 to 32, LCDR2 comprises amino acid residues 50 to 52, LCDR3 comprises amino acid residues 89 to 97, HCDR1 comprises amino acid residues 26 to 33, HCDR2 comprises amino acid residues 51 to 57 and HCDR3 comprises amino acid residues 93 to 102. Residues numbers for the Kabat, Chothia and IMGT CDRs are given as Kabat position numbers.

[0105]Exemplary methods for characterization of amino acid residues in an antibody binding domain may include: a determination of each amino acid residue's proximity group as designated on the "prox" line of FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D and additionally or alternatively a determination of each amino acid residue's conspicuousness as calculated by the methods provided in the present disclosure.

[0106]A. Determination of Proximity Groups

[0107]The characterization process may determine the proximity group for each amino-acid position simply by inspecting the corresponding symbol ("CPSI.:") on the "prox" lines as shown, for example, in FIG. 2A, 2B, 2C and/or 2D. In some embodiments, the antibody's light-chain and/or heavy-chain sequences are aligned with appropriate sequences (e.g., such as consensus or germline sequences) and also with the "prox" lines of the present methods (FIGS. 2A, 2B, 2C and/or 2D),

[0108]Each position in the light chain and heavy chain has been assigned to one of five novel groups designated as contacting (C), peripheral (P), supporting (S), interfacial (I), or distant (D) on the "prox" lines, for example, of FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D according to the methods disclosed herein. These Figures (e.g., 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D) contain a disc line to reflect disconnection from any significant effect upon an antibody's binding site, and an outw line to reflect outward-orientation on an antibody's surface.

[0109]Table 1 shows five proximity groups, as well as a novel designation of disconnection (as shown on a "disc" line, for example, in FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D) and outward-orientation (shown as an "outw" line, for example, in FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D) as defined for each group. The number of positions of each type of proximity group for an exemplary antibody (e.g., ING-1, as described herein) in a light chain, a heavy chain, and both chains together are shown in Table 2.

TABLE-US-00001 TABLE 1 Proximity Abbr Disc/Outw Contacting C -+ -o Peripheral P o+ oo Supporting S -- o- Interfacial I -= o= += Distant • ++ +o +- p c

TABLE-US-00002 TABLE 2 Proximity L H L + H Contacting 16 21 37 Peripheral 3 8 11 Supporting 14 16 30 Interfacial 9 10 19 Distant 70 63 133

[0110]Without being bound by a theory of the invention, it has been hypothesized that amino acid residues designated as contacting (C) are located within the combining site (see, e.g., "-" on the "disc" line of FIG. 2A, 2B, 2C and/or 2D), and their sidechains are mostly outward-oriented (see, e.g., "+" or "o" on the outw line). It has been further hypothesized that these are generally surface-exposed residues in the CDR loops themselves, so their sidechains are very favorably situated for making direct contact with corresponding residues on a binding partner.

[0111]Without being bound by a theory of the invention, it has been hypothesized that amino acid residues designated as peripheral (P) are slightly disconnected from the binding site (see, e.g., "o" on the "disc" line), and their sidechains are mostly outward-oriented (see, e.g., "+" or "o" on the outw line). Many of these are framework residues with variable orientation, which are located at curves or twists in the polypeptide chain not too far from CDR loops. Although they may normally not make direct contact with a binding partner, they may possibly make contact if a particular binding partner is bound preferentially toward one side of the binding site instead of being centered.

[0112]Without being bound by a theory of the invention, it has been hypothesized that amino acid residues designated as supporting (S) are either directly within or close to the combining site (see, e.g., "-" or "o" on the "disc" line), and their sidechains are inward-oriented, e.g., "-" on the outw line). It has been further hypothesized that many of these residues are buried in the Vernier-zone platform directly underneath a combining site, so that their nonpolar sidechains are able to act as conformation-stabilizing "anchors" for CDR loops which rest on top of them.

[0113]Without being bound by a theory of the invention, it has been hypothesized that amino acid residues designated as interfacial (I) may be located anywhere in relation to the binding site (see, e.g., "+" or "o" or "-" on the "disc" line), but their sidechains form the interface between the light and heavy subunits of the variable domain (see, e.g., "_" on the outw line). It has been further hypothesized that amino acid changes of these residues may cause the two subunits to pivot or rotate relative to one another along their shared hydrophobic interfacial surface, producing strong allosteric effects upon an entire binding site, for example, all six CDR loops may be forced to change their conformation in response.

[0114]Without being bound by a theory of the invention, it has been hypothesized that amino acid residues designated as distant (D) are of two different types, with those of the first type being disconnected from a combining site and its targeted epitope (see, e.g., "+" on the "disc" line), and their sidechains may have any orientation except interfacial (see, e.g., "+" or "o" or "-" but not "=" on the outw line). It is further hypothesized that amino acid changes at these positions generally will have little or no effect on enhanced affinity to a binding partner.

[0115]B. Determination of Conspicuousness

[0116]In some embodiments, alternatively or additionally with determination of the proximity groups by inspection of the "prox" lines, the characterization process may involve a calculation of the conspicuousness value for each amino-acid position. The conspicuousness value of a sidechain at a particular antibody position is hypothesized to represent the degree to which it appears strikingly different or unusually outstanding in comparison with selected sequences (e.g., a consensus or germline sequence). Without being bound by a theory of the invention, this value indicates the likelihood that this particular residue may be a somatic mutation which was necessary to confer binding partner specific affinity upon an antibody. Consequently, the conspicuousness value also correlates with the hypothesis that a new engineered amino acid substitution at or near this position could possibly lead to forming or strengthening a bond with a residue on a binding partner surface.

[0117]Conspicuousness values are calculated by comparing each sidechain of a candidate antibody with the corresponding sidechain of an appropriate consensus or germline sequence, for example, from a mutual alignment. For example, numerical values for conspicuousness can be calculated readily for each amino-acid position in a given antibody, according to the following formula: add 1 point for each three units of difference in size (e.g., divide the absolute value of the size-difference by 3 and drop the decimal without rounding); add 1 point for a shift from one sidechain class to another; add 1 point for each unit (absolute value) of difference in charge, and add 1 point for nonidentity (see, e.g., FIG. 4).

[0118]For example, where a single antibody sequence is aligned or compared with a single consensus or germline sequence, there is one "pair" of sequences being compared. The conspicuousness value for each amino-acid position in the alignment or comparison is the sum of the points for chemical function and physical size and nonidentity at that position. Where more than two sequences are aligned or compared together at the same time, each of the antibody sequences may form a separate "pair" with each of the consensus or germline sequences. The conspicuousness values are calculated as described (e.g., sum of function and size and nonidentity) for each pair of sequences being aligned or compared, and then the overall conspicuousness value for each amino acid position in the whole alignment is the sum of the values obtained from each pair at that position, while also adding in a value for repeated identical mutations.

[0119]It is hypothesized that nonidentity simply marks an amino-acid position as minimally conspicuous if it displays any kind of difference when compared with a corresponding consensus or germline position. Even a conservative mutation (e.g., from leucine to isoleucine or valine) may suggest a possible bond with a binding partner, especially if a slight change of size or shape was necessary to fine-tune steric relationships between the two molecules.

[0120]An exemplary calculation of conspicuousness is illustrated as follows. Four monoclonal antibodies to the same epitope were isolated, and portions of their heavy chains were mutually aligned with a germline sequence, between Kabat positions 25 and 57 [Mendez et al., Nature Genetics, 15:146-152 (1997)] (see, Table 3). Since this alignment contains more than two sequences, each of the four antibody sequences can separately form a "pair" with the one germline sequence. Thus, conspicuousness values are calculated separately for each of the four pairs, and then totaled at each amino-acid position, while also adding in the additional values for repeated identical mutations.

TABLE-US-00003 TABLE 3 prox: PSSCSCCCCSISI.I.:...I.ISSCSCCCCCC pos: 30 40 50 germ: GSISSGGYYWSWIRQHPGKGLEWIGYIYYSGST mAb1: N D S N mAb2: D T N mAb3: v D p HL N mAb4: N D DC

[0121]Three repetitions are shown in Table 3, at positions 28 and 31 and 56. In each of these cases, an identical amino acid (N or D) has appeared at the same location in more than one independently isolated antibody. Accordingly, as described herein, these positions are given very high conspicuousness in the affinity enhancement process. An additional 2 points are added for each repetition of an identical amino acid at a given position (e.g., four D's amount to three repetitions of the first D, so it is worth 3×2=6 points).

[0122]In an example, at position 50, the first pair (germ:mAb 1) gets 3 points (Y to S=2 for size+0 for class+0 for charge+1 for nonidentity), the second pair (germ:mAb2) gets 0 points (unmutated Y=0+0+0+0), the third pair (germ:mAb3) gets 3 points (Y to H=0 for size+1 for class+1 for charge+1 for nonidentity), and the fourth pair (germ:mAb4) gets 0 points (unmutated Y=0+0+0+0). The total conspicuousness for position 50 is the sum (3+0+3+0) of these, plus 0 extra points for no repeated identical mutations, which finally gives 6.

[0123]In another example, at position 28, the first pair gets 1 point (S to N=0+0+0+1), the second and third pairs get 0 points, and the fourth pair gets I point. Since the somatic mutation N appears at position 28 twice, it is repeated once, and thus gets 2 extra points. The total conspicuousness for position 28 is the sum (1+0+0+1), plus 2 points for one repetition, which finally gives 4.

[0124]In another example, at position 31, each of the four pairs gets 4 points (G to D=1+1+1+1). Since the somatic mutation D appears at position 31 four times, it is repeated three times, and thus gets 3×2=6 extra points. The total conspicuousness for position 28 is the sum (4+4+4+4), plus 6 points for three repetitions, which finally gives 22.

[0125]The conspicuousness points can be calculated (one pair at a time and then summed) for positions 28, 31, and 50 in the antibody sequence provided in Table 2.

Methods for Targeted Affinity Enhancement

[0126]The present disclosure provides methods for the change of an amino acid residue at a position in an antibody variable domain with other amino acid residues to identify an amino acid change which results in the antibody variable domain having enhanced binding affinity for its binding partner. Enhanced binding affinity refers to a modified variable domain that binds to a binding partner (e.g., antigen) with a significantly higher equilibrium constant of association (K_A) or lower equilibrium constant of dissociation (K_D) than the parent variable domain when the amounts of modified and parent variable domains in the binding assay are the same. For example, the modified variable domain with improved binding affinity may display at least 10%, at least 15%, at least 25%, at least 50%, at least 75%, at least 100% (or two-fold), at least 5-fold, at least 8-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more, higher affinity to a binding partner than the corresponding parent variable domain. As used herein, binding partner refers to an antigen (e.g., an epitope on an antigen) recognized by an antibody or a molecular target of an antibody.

[0127]Amino acid residues in an antibody variable domain that are likely to contribute to an antibody's binding affinity to a binding partner may be changed to other amino acid residues to determine which change results in an enhancement of binding affinity. These residues may be changed with other amino acid residues to generate a library or array of modified variable domains which may be selected for enhanced binding affinity to a binding partner as compared to the unmodified (parent) variable domain. These residues preferably include: alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Cysteine and methionine may be included but are not preferred. In some embodiments cysteine and methionine are specifically excluded.

[0128]In some embodiments, methods for targeted affinity enhancement may utilize an amino acid residue's proximity group and/or conspicuousness as described above, to select for those amino acid positions where an amino acid change is likely to enhance binding affinity.

[0129]An exemplary method for targeted affinity enhancement includes aligning monoclonal antibody sequences with consensus or individual light-chain and heavy-chain sequences according to a standard numbering system such as Kabat; optionally co-aligning with the antibody's own direct germline precursor sequences if they are known; optionally characterizing each antibody position based upon the degree to which the residue differs from the corresponding consensus or germline residue in terms of charge or size or chemical functionality; preferably changing one or more contacting (C), supporting (S), peripheral (P) and/or interfacial (I) amino acid residues with other amino acids residues to produce a library or array of modified variable domains; and selecting those modified variable domains that have enhanced affinity to a binding partner compared to the unmodified variable domain. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

Methods of Making Antibody Variable Domains With Enhanced Binding Affinity

[0130]Methods are provided for enhancing the binding affinity of an antibody by means of producing targeted amino acid changes in the antibody's variable domain. For example, engineered amino acid changes are introduced at positions likely to produce enhanced affinity based upon an amino acid residue's proximity group.

[0131]In an exemplary method, amino acid changes are engineered at one or more amino acid residues categorized as preferably contacting (C), peripheral (P), supporting (S) and/or interfacial on the "prox" lines of FIG. 2A, 2B, 3A, 3B, 3C and/or 3D. In other embodiments, amino acid residues categorized in more than one group may be selected for change. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0132]For example, methods are provided for making a modified variable domain of an antibody with enhanced binding affinity by modifying the nucleotide sequence of an antibody variable domain at a position that preferably encodes a contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino acid residue identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D, thereby generating a modified antibody variable domain; and selecting a modified variable domain that has enhanced binding affinity. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0133]Methods are also provided for generating an array of modified antibody variable domains with eighteen amino acid changes at one or more contacting (C) residues from a collection of modified variable domains by obtaining a collection of modified antibody variable domains containing amino acid changes at one or more contacting (C) residues; sequencing the collection of modified variable domains; and arranging each sequenced modified antibody variable domain comprising one of the eighteen amino acid changes at one or more contacting (C) residue to generate an array of modified variable domains with eighteen amino acid changes at one or more contacting (C) residues.

[0134]Methods are provided for generating an array of modified variable domains with eighteen amino acid changes at one or more contacting (C) residues by (a) synthesizing polynucleotides that encode sequences that vary at one or more contacting (C) residues and contain eighteen amino acid changes at each contacting (C) residue to generate modified antibody variable domains; and (b) arranging each synthesized polynucleotide from step (a) to generate an array of synthesized polynucleotides with eighteen amino acid changes at one or more contacting (C) residues.

[0135]Methods are provided for generating an array of modified variable domains with eighteen amino acid changes at one or more contacting (C) residues by (a) synthesizing polynucleotides that encode sequences that vary at one or more contacting (C) residues and contain eighteen amino acid changes at each contacting (C) residue to generate modified antibody variable domains; (b) transfecting each synthesized polynucleotide of step (a) separately into a host cell to generate clones comprising the synthesized polynucleotides; and (c) arranging each clone from step (b) to generate an array of clones capable of expressing modified variable domains with eighteen amino acid changes at one or more contacting (C) residues.

[0136]In some embodiments, one or more contacting residues to be changed may be in complementarity determining domain-1 (CDR1) in a light chain variable domain. In certain embodiments, the contacting residues may be at a position corresponding to position 28, 30 and/or 31 in CDR1.

[0137]In other embodiments, one or more contacting (C) residues to be changed may be in CDR2 in a light chain variable domain. In certain embodiments, the contacting (C) residues may be at a position corresponding to position 50, 51 and/or 53 in CDR2.

[0138]In some embodiments, one or more contacting (C) residues to be changed may be in CDR1 in a heavy chain variable domain. In certain embodiments, the contacting (C) residues may be at a position corresponding to position 32 and/or 33 in CDR1.

[0139]In some embodiments, one or more (C) contacting residues to be changed may be in CDR2 in a heavy chain variable domain. In certain embodiments, the contacting (C) residues may be at a position corresponding to position 50, 52, 53, 54, 56, and/or 58 in CDR2.

[0140]Modified variable domains are synthesized by modifying the nucleic acid of a parent variable domain, inserting the modified nucleic acid into an appropriate cloning vector and expressing the modified nucleic acid to produce modified variable domains. Exemplary protocols are described below.

[0141]1. Making Modified Variable Domain Nucleic Acids

[0142]Modified variable domains comprise one or more amino acid sequence changes (e.g., substitutions) relative to a parent variable domain sequence to provide for enhanced binding affinity to a binding partner compared to the parent variable domain.

[0143]In some embodiments, modified variable domains may have one or more amino acid changes at preferably a contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino acid residue identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed. In some embodiments, a library of modified variable domains may be constructed comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid changes at preferably a contacting (C), peripheral (P), supporting (S), interfacial (I) residue and/or less preferably at a distant (D) amino acid residue identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D.

[0144]In some embodiments, an amino acid residue at preferably one or more contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino acid residues, as identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D, may be changed with one or more of the following preferred amino acid residues: alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed. In other embodiments, an amino acid residue at preferably a contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino acid residue, as identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D is changed with all of the following amino acid residues: alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0145]In some embodiments, a modified variable domain may have two or more amino acid changes at preferably a contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino residue identified from the "prox" line as shown in FIG. 3A, 3B, 3C and/or 3D. Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0146]In some embodiments, a modified variable domain exhibits enhanced binding affinity to a binding partner compared to the binding affinity exhibited by the parent variable domain. In some embodiments, a modified variable domain exhibits at least a 10%, at least a 15%, at least a 25%, at least a 50%, at least a 75%, at least a 100% (or a two-fold), at least a 5-fold, at least an 8-fold, at least a 10-fold, at least a 50-fold, at least a 100-fold, or more, higher affinity to a binding partner than the corresponding parent variable domain.

[0147]A library and/or an array of modified variable domains may be generated which contain multiple amino acid changes at a position of interest (e.g., at an amino acid residue in an antibody's variable domain at preferably a contacting (C), peripheral (P), supporting (S) and/or interfacial (I) amino acid residue as designated on the "prox" line of FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D). Less preferably one or more distant (D) amino acid residues may additionally or alternatively be changed.

[0148]For example, amino acids may be preferably incorporated into a position of interest by utilizing from three to nine different degenerate codons. Each degenerate codon will produce a mixture of from two to twelve different oligonucleotides. One example of a seven degenerate primer set produces basic amino acid changes\with a single primer that contains the degenerate codon of ARG (R=A/G), encoding Arg/Lys. Polar amino acid changes can be produced with two primers. For example, the first primer contains the degenerate codon WMC (W=A/T; M=A/C), encoding Ser/Thr/Asn/Tyr, while the second polar primer utilizes the degenerate codon CAS (S=C/G), encoding His/Gln. Acidic amino acid changes can be produced with a single degenerate codon of GAS, encoding Glu/Asp. Non-polar functional amino acid changes can be produced with three primers: NTC (N=A/G/C/T), encoding Leu/Phe/Ile/Val, KGG (K=G/T), encoding Trp/Gly, and SCG, encoding Pro/Ala

[0149]An alternate substitution method may employ the use of three primers each comprising a different degenerate codon to produce eighteen amino acid changes. For example, the codons may include: NHT (where N=A/G/C/T, H=A/C/T), which codes for Phe/Ser/Tyr/Leu/Pro/His/Ile/Thr/Asn/Val/Ala/Asp; VAA (where V=A/C/G), which codes for Gln/Lys/Glu; and BGG (where B=C,G,T), which codes for Trp/Arg/Gly.

[0150]An alternate substitution method also may employ a nine degenerate primer set by producing basic amino acid changes \with a single primer that contains the degenerate codon of ARG (R=A/G), encoding Arg/Lys. Polar amino acid changes can be produced with three primers. For example, the first primer contains the degenerate codon WAC (W=A/T; M=A/C), encoding Asn/Tyr, while the second polar primer utilizes the degenerate codon WCC, encoding Ser/Thr, while the third polar primer utilizes CAS (S=C/G), encoding His/Gln. Acidic amino acid changes can be produced with a single degenerate codon of GAS, encoding Glu/Asp. Non-polar functional amino acid changes can be produced with five primer sets: MTC (M=A/C), encoding Leu/Ile, KTC (K=G/T) encoding PheNal, KGG (K=G/T), encoding Trp/Gly, and SCG, encoding Pro/Alaln some embodiments, all seven of the degenerate primers are used to perform one PCR reaction. In other embodiments, each degenerate primer is used in a separate PCR reaction. Any combination of PCR primers may be used in a PCR reaction.

[0151]DNA encoding modified variable domains may be prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by primer-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared modified variable domain or parent variable domain. These techniques may utilize antibody nucleic acid (DNA or RNA), or nucleic acid complementary to the antibody nucleic acid.

[0152]DNA encoding a modified variable domain with more than one amino acid to be changed may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one primer that codes for all of the desired amino acid changes. If, however, the amino acids are located some distance from each other (separated by more than about ten amino adds), it is more difficult to generate a single primer that encodes all of the desired changes. Instead, one of two alternative methods may be employed.

[0153]In the first method, a separate primer is generated for each amino acid to be changed. The primers are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid changes.

[0154]The alternative method involves two or more rounds of mutagenesis to produce the desired mutant antibody. The first round is as described for the modified variable domain which comprise one amino acid change: wild-type DNA is used for the template, a primer encoding the first desired amino acid change(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The primer encoding the additional desired amino acid change(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.

[0155]2. Insertion of DNA into a Cloning Vehicle

[0156]The cDNA or genomic DNA encoding the modified antibody variable domain may be inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Many vectors are available, and selection of the appropriate vector will depend on 1) whether it is to be used for DNA amplification or for DNA expression, 2) the size of the DNA to be inserted into the vector, and 3) the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0157]For example, the cDNA or genomic DNA encoding the modified variable domain may be inserted into a modified phage vector (i.e. phagemid). Construction of phage display libraries exploits the bacteriophage's ability to display peptides and proteins on their surfaces, i.e., on their capsids. Often, filamentous phage such as M13, f1 or fd are used. Filamentous phage contain single-stranded DNA surrounded by multiple copies of genes encoding major and minor coat proteins, e.g., pIII. Coat proteins are displayed on the capsid's outer surface. DNA sequences inserted in-frame with capsid protein genes are co-transcribed to generate fusion proteins or protein fragments displayed on the phage surface. Peptide phage libraries thus can display peptides representative of the diversity of the inserted genomic sequences. Significantly, these epitopes can be displayed in "natural" folded conformations. The peptides expressed on phage display libraries can then bind target molecules, i.e., they can specifically interact with binding partner molecules such as antibodies (Petersen (1995) Mol. Gen. Genet. 249:425-31), cell surface receptors (Kay (1993) Gene 128:59-65), and extracellular and intracellular proteins (Gram (1993) J. Immunol. Methods 161:169-76).

[0158]The concept of using filamentous phages, such as M13, fd or fl, for displaying peptides on phage capsid surfaces was first introduced by Smith (1985) Science 228:1315-1317. Peptides have been displayed on phage surfaces to identify many potential ligands (see, e.g., Cwirla (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382). There are numerous systems and methods for generating phage display libraries described in the scientific and patent literature (see, e.g., Sambrook and Russell, Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Chapter 18, 2001; "Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, 1996; Crameri (1994) Eur. J. Biochem. 226:53-58; de Kruif (1995) Proc. Natl. Acad. Sci. USA 92:3938-42; McGregor (1996) Mol. Biotechnol. 6:155-162; Jacobsson (1996) Biotechniques 20:1070-1076; Jespers (1996) Gene 173:179-181; Jacobsson (1997) Microbiol Res. 152:121-128; Fack (1997) J. Immunol. Methods 206:43-52; Rossenu (1997) J. Protein Chem. 16:499-503; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45; Rader (1997) Curr. Opin. Biotechnol. 8:503-508; Griffiths (1998) Curr. Opin. Biotechnol. 9:102-108).

[0159]Typically, exogenous nucleic acid to be displayed are inserted into a coat protein gene, e.g. gene III or gene VIII of the phage. The resultant fusion proteins are displayed on the surface of the capsid. Protein VIII is present in approximately 2700 copies per phage, compared to 3 to 5 copies for protein III (Jacobsson (1996), supra). Multivalent expression vectors, such as phagemids, can be used for manipulation of exogenous genomic or antibody encoding inserts and production of phage particles in bacteria (see, e.g., Felici (1991) J. Mol. Biol. 222:301-310).

[0160]Phagemid vectors are often employed for constructing the phage library. These vectors include the origin of DNA replication from the genome of a single-stranded filamentous bacteriophage, e.g., M13, f1 or fd. A phagemid can be used in the same way as an orthodox plasmid vector, but can also be used to produce filamentous bacteriophage particle that contain single-stranded copies of cloned segments of DNA.

[0161]Other phage can also be used. For example, T7 vectors can be employed in which the displayed product on the mature phage particle is released by cell lysis.

[0162]In addition to phage epitope display libraries, analogous epitope display libraries can also be used. For example, the methods of the disclosure can also use yeast surface displayed epitope libraries (see, e.g., Boder (1997) Nat. Biotechnol. 15:553-557), which can be constructed using such vectors as the pYD1 yeast expression vector. Other potential display systems include mammalian display vectors and E. coli libraries.

[0163]An antibody or antibody fragment, e.g., a scFv, Fab or Fv may be displayed on the surface of a phage using phage display techniques. Exemplary antibody phage display methods are known to those skilled in the art and are described, e.g., in Hoogenboom, Overview of Antibody Phage-Display Technology and Its Applications, from Methods in Molecular Biology: Antibody Phage Display: Methods and Protocols (2002) 178:1-37 (O'Brien and Aitken, eds., Human Press, Totowa, N.J.). For example, a library or array of antibodies or antibody fragments (e.g., scFvs, Fabs, Fvs with an engineered intermolecular disulfide bond to stabilize the V_H-V_L pair, and diabodies) can be displayed on the surface of a filamentous phage, such as the nonlytic filamentous phage fd or M13. Antibodies or antibody fragments with the desired binding specificity can then be selected.

[0164]An antibody phage-display library can be prepared using methods known to those skilled in the art (see, e.g., Hoogenboom, Overview of Antibody Phage-Display Technology and Its Applications, from Methods in Molecular Biology: Antibody Phage Display: Methods and Protocols (2002) 178:1-37 (O'Brien and Aitken, eds., Human Press, Totowa, N.J.).

[0165]In some embodiments, cDNA is cloned into a phage display vector, such as a phagemid vector (e.g., pCES1, p XOMA Fab or pXOMA Fab-gIII). In certain embodiments, cDNA encoding both heavy and light chains may be present on the same vector. In some embodiments, cDNA encoding scFvs are cloned in frame with all or a portion of gene III, which encodes the minor phage coat protein pIII. The phagemid directs the expression of the scFv-pIII fusion on the phage surface. In other embodiments, cDNA encoding heavy chain (or light chain) may be cloned in frame with all or a portion of gene III, and cDNA encoding light chain (or heavy chain) is cloned downstream of a signal sequence in the same vector. The signal sequence directs expression of the light chain (or heavy chain) into the periplasm of the host cell, where the heavy and light chains assemble into Fab fragments. Alternatively, in certain embodiments, cDNA encoding heavy chain and cDNA encoding light chain may be present on separate vectors. In certain embodiments, heavy chain and light chain cDNA may be cloned separately, one into a phagemid and the other into a phage vector, which both contain signals for in vivo recombination in the host cell.

[0166]The techniques for constructing and analyzing phage display libraries uses recombinant technology well known to those of skill in the art. General techniques, e.g., manipulation of nucleic encoding libraries, epitopes, antibodies, and vectors of interest, generating libraries, subcloning into expression vectors, labeling probes, sequencing DNA, DNA hybridization are described in the scientific and patent literature, see e.g., Sambrook and Russell, eds., Molecular Cloning: a Laboratory Manual (3rd), Vols. 1-3, Cold Spring Harbor Laboratory Press, (2001); Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York (1997-2001) ("Ausubel"); and, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993). Sequencing methods typically use dideoxy sequencing, however, other methodologies are available and well known to those of skill in the art.

[0167]3. Transformation of Host Cells

[0168]Suitable host cells for cloning or expressing the vectors herein may include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli, Bacilli such as B. subtilis, Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescens.

[0169]For example, recombinant phagemid or phage vectors may be introduced into a suitable bacterial host, such as E. coli. In some embodiments using phagemid, the host may be infected with helper phage to supply phage structural proteins, thereby allowing expression of phage particles carrying the antibody-pIII fusion protein on the phage surface.

Methods for Identifying an Antibody Variable Domain Having Enhanced Affinity for a Binding Partner

[0170]Methods are provided for identifying a modified antibody variable domain having enhanced binding affinity for a binding partner by contacting a parent antibody variable domain with the binding partner under conditions that permit binding; contacting modified antibody variable domains made by the methods of the present disclosure with the binding partner under conditions that permit binding; and determining binding affinity of the modified antibody variable domains and the parent antibody variable domain for the binding partner, wherein modified antibody variable domains that have a binding affinity for the binding partner greater than the binding affinity of the parent antibody variable domain for the binding partner are identified as having enhanced binding affinity.

[0171]Isolated antibody variable domains may exhibit binding affinity as single chains, in the absence of assembly into a heteromeric structure with their respective V_H or V_L subunits. As such, populations of V_H and V_L altered antibody variable domains can be expressed alone and screened for binding affinity having substantially the same or greater binding affinity compared to the parent antibody V_H or V_L variable domain.

[0172]Alternatively, populations of antibody V_H and V_L altered variable domains polypeptides can be co-expressed so that they self-assemble into heteromeric altered antibody variable domain binding fragments. The heteromeric binding fragment population can then be screened for species exhibiting enhanced binding affinity to a binding partner compared to the binding affinity of the parent antibody variable domain.

[0173]The expressed population of modified antibody variable domains can be screened for the identification of one or more altered antibody variable domain species which exhibit enhanced binding affinity to a binding partner as compared with the parent antibody variable domain. Screening can be accomplished using various methods well known in the art for determining the binding affinity of a polypeptide or compound. Additionally, methods based on determining the relative affinity of binding molecules to their partner by comparing the amount of binding between the modified antibody variable domain and the binding partner can similarly be used for the identification of species exhibiting binding affinity substantially the same or greater than the parent antibody variable domain to the binding partner. The above methods can be performed, for example, in solution or in solid phase. Moreover, various formats of binding assays are well known in the art and include, for example, immobilization to filters such as nylon or nitrocellulose; two-dimensional arrays, enzyme linked immunosorbant assay (ELISA), radioimmuno-assay (RIA), panning and plasmon resonance (see, e.g., Sambrook et al., supra, and Ansubel et al., supra).

[0174]For the screening of populations of polypeptides such as the modified antibody variable domains produced by the methods of the disclosure, immobilization of the modified antibody variable domains to filters or other solid substrates is particularly advantageous because large numbers of different species can be efficiently screened for binding to a binding partner. Such filter lifts allow for the identification of modified antibody variable domains that exhibit enhanced binding affinity compared to the parent antibody variable domain to the binding partner. Alternatively, the modified antibody variable domains may be expressed on the surface of a cell or bacteriophage. For example, panning on an immobilized binding partner can be used to efficiently screen for the relative binding affinity of species within the population of modified antibody variable domains and for those which exhibit enhanced binding affinity to the binding partner than the parent antibody variable domain.

[0175]Another affinity method for screening populations of modified antibody variable domains is a capture lift assay that is useful for identifying a binding molecule having selective affinity for a ligand. This method employs the selective immobilization of modified antibody variable domains to a solid support and then screening of the selectively immobilized modified antibody variable domains for selective binding interactions against the binding partner. Selective immobilization functions to increase the sensitivity of the binding interaction being measured since initial immobilization of a population of modified antibody variable domains onto a solid support reduces non-specific binding interactions with irrelevant molecules or contaminants which can be present in the reaction.

[0176]Another method for screening populations or for measuring the affinity of individual modified antibody variable domains is through surface plasmon resonance (SPR). This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal/liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal. The binding event can be either binding association or disassociation between a receptor-ligand pair. The changes in refractive index can be measured essentially instantaneously and therefore allows for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (k_on) and disassociation rates (k_off).

[0177]Measurements of k_on and k_off values can be advantageous because they can identify modified antibody variable domains with enhanced binding affinity for a binding partner. For example, a modified antibody variable domain can be more efficacious because it has, for example, a higher k_on valued compared to the parent antibody variable domain. Increased efficacy is conferred because molecules with higher k_on values can specifically bind and inhibit their binding partner at a faster rate. Similarly, a modified antibody variable domain can be more efficacious because it exhibits a lower k_off value compared to molecules having similar binding affinity. Increased efficacy observed with molecules having lower k_off rates can be observed because, once bound, the molecules are slower to dissociate from their binding partner.

[0178]Methods for measuring the affinity, including association and disassociation rates using surface plasmon resonance are well known in the arts and can be found described in, for example, Jonsson and Malmquist, Advances in Biosensors, 2:291-336 (1992) and Wu et al. Proc. Natl. Acad. Sci. USA, 95:6037-6042 (1998).

[0179]Using any of the above described screening methods, a modified antibody variable domain having binding affinity substantially the same or greater than the parent variable domain is identified by detecting the binding of at least one altered variable domain within the population to its binding partner.

[0180]Detection methods for identification of species within the population of modified variable domains can be direct or indirect and can include, for example, the measurement of light emission, radioisotopes, calorimetric dyes and fluorochromes. Direct detection includes methods that operate without intermediates or secondary measuring procedures to assess the amount of the binding partner bound by the modified antibody variable domain. Such methods generally employ ligands that are themselves labeled by, for example, radioactive, light emitting or fluorescent moieties. In contrast, indirect detection includes methods that operate through an intermediate or secondary measuring procedure. These methods generally employ molecules that specifically react with the binding partner and can themselves be directly labeled or detected by a secondary reagent. For example, a modified antibody variable domain specific for a binding partner can be detected using an antibody capable of interacting with the modified antibody variable domain, again using the detection methods described above for direct detection. Indirect methods can additionally employ detection by enzymatic labels. Moreover, for the specific example of screening for catalytic antibodies, the disappearance of a substrate or the appearance of a product can be used as an indirect measure of binding affinity or catalytic activity.

[0181]In some embodiments, the modified antibody variable domain has a binding affinity for the binding partner greater than the binding affinity of the parent variable domain for the binding partner and thus is identified as having enhanced binding affinity.

[0182]In some embodiments, a modified antibody variable domain exhibits enhanced binding affinity to a binding partner compared to the binding affinity between the parent variable domain and the binding partner. In some embodiments, a modified variable domain exhibits an at least 10%, at least 15%, at least 25%, at least 50%, at least 75%, at least 100% (or two-fold), at least 5-fold, at least 8-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more, higher affinity to a binding partner than the corresponding parent antibody variable domain.

[0183]In other embodiments, the modified antibody variable domain has a binding affinity for the binding partner less than the binding affinity of the parent antibody variable domain for the binding partner and thus is identified as having reduced binding affinity for the binding partner.

[0184]This disclosure is further illustrated by the following examples which are provided to facilitate the practice of the disclosed methods. These examples are not intended to limit the scope of the disclosure in any way.

Examples

Example 1

Design of Primers for Synthesis of Nucleic Acid Encoding Modified Antibody Variable Domains

[0185]Each contacting residue identified from the "prox" lines in FIG. 2, 3A, 3B, 3C and/or 3D may be changed with other amino acid residues (e.g., alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) by performing PCR with an oligonucleotide containing one of seven different degenerate codons (e.g., ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G)).

[0186]In an exemplary substitution method, use of seven primers, each comprising one of the seven degenerate codons, may be employed to change one or more contacting (C) amino acid positions in a parent nucleic acid molecule to 18 other amino acid residues. An alternate substitution method may employ the use of three primers each comprising a different degenerate codon to produce eighteen amino acid changes at one or more contacting resides in a parent nucleic acid molecule. For example, the codons may include: NHT (where N=A/G/C/T, H=A/C/T), which codes for Phe/Ser/Tyr/Leu/Pro/His/Ile/Thr/Asn/Val/Ala/Asp; VAA (where V=A/C/G), which codes for Gln/Lys/Glu; and BGG (where B=C,G,T), which codes for Trp/Arg/Gly. This allows production of all eighteen amino acids at equal ratios if oligonucleotides comprising NHT is used at a 4:1:1 ratio with oligonucleotides comprising VAA and oligonucleotides comprising BGG, since NHT encodes twelve amino acids and VAA and BGG both encode three amino acids.

[0187]Primers containing one or more degenerate codons may be used to introduce a desired class of amino acid residue at a contacting (C) position by hybridizing to a parent nucleic acid (e.g., the nucleotide sequence encoding the degenerate codon pairs with a contacting (C) position to be changed). Basic amino acid changes can be produced at a contacting (C) position with a single oligonucleotide that contains the codon mixture of ARG (R=A/G), encoding Arg/Lys. Further, polar amino acid changes can be introduced at a contacting (C) position with two oligonucleotides. The first oligonucleotide contains the codon mixture WMC (W=A/T; M=A/C), encoding Ser/Thr/Asn/Tyr, while the second oligonucleotide utilizes the codon mixture CAS (S=C/G), encoding His/Gln. Additionally, acidic amino acid changes may be introduced at a contacting (C) position with a single codon mixture of GAS, encoding Glu/Asp. Last, non-polar amino acid changes may be introduced at a contacting (C) position with a mixture of three primers with degenerate codons: NTC (N=A/G/C/T), encoding Leu/Phe/Ile/Val, KGG (K=G/T), encoding Trp/Gly, and SCG, encoding Pro/Ala.

Example 2

Construction of a Library Containing Modified Antibody Variable Domains

[0188]Modified antibody variable domains containing amino acid changes at one or more contacting (C) residues present within an exemplary antibody, for example, ING-1 (a mouse-human chimeric antibody containing the Br-1 mouse variable region domains and human constant regions domains which selectively binds to Ep-CAM (U.S. Pat. No. 5,576,184), heavy chain sequence represented by SEQ ID NO: 579, light chain sequence represented by SEQ ID NO: 580) may be synthesized by PCR amplification from a parent nucleic acid molecule using synthetic oligonucleotides containing a degenerate codon (SEQ ID NO: 1-285 or SEQ ID NO: 583-699). Similarly, modified antibody variable domains containing amino acid changes at one or more contacting (C) residues present within an exemplary antibody, for example, IL-1 antibody (heavy chain sequence represented by SEQ ID NO: 581, kappa chain sequence represented by SEQ ID NO: 582) may be synthesized by PCR amplification from a parent nucleic acid molecule using synthetic oligonucleotides containing a degenerate codon (SEQ ID NO: 286-578 or SEQ ID NO: 700-806).

[0189]For example, each library primer containing the degenerate codon described above for ING-1 may be used in a PCR reaction to synthesize a DNA fragment which incorporates an amino acid change and a 3' restriction site. In an exemplary method, PCR may be conducted at a contacting (C) position (e.g., H3-3) by utilizing the CDRH3 oligonucleotide H3-3NP2 (SEQ ID NO: 267): 5'-GCTACATATTTCTGTGCAAGATTTGGCTCTKGGGTGGACTACTGGGGTCAAG G-3', and the reverse primer Notl-R (SEQ ID NO: 285): 5'-AGCGGCCGCACAAGATTTGGGCTCAACTCTC-3') (see, FIG. 5) under standard conditions (see, e.g., Sambrook and Russell, Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001). After PCR amplification, fragments are obtained which comprise either a tryptophan or glycine residue at the internal codon (underlined above). Further, six other PCR reactions may be performed at the H3-3 position, utilizing SEQ ID NO: 285 with one of SEQ ID NOs: 262-266 and 268 under the conditions described above to obtain other amino acid changes at the site. Next, the products from these reactions may be combined at equal mass, except for reactions which used SEQ ID NO: 263 and 266 as a primer (this mixture is termed the pooled H3-3 library). Due to the degeneracy of these primers, twice the mass of the sample obtained with SEQ ID NO: 263 and 266 is added to produce an equimolar ratio of encoded amino acids.

[0190]An additional PCR reaction may be performed to create a fragment (called the H3-R fragment) which contains a 5' restriction site and an overlapping complementary region to the library fragments described above. As an example, for the H3-3 position, a PCR reaction may be performed utilizing the Asc-F2 (SEQ ID NO: 284) and one of the H3R (SEQ ID NO: 247) primer. The 3' portion of this molecule contains a region that is identical to the 5' portion of the molecules created above which permits the use of a PCR reaction to create a contiguous molecule containing a 5' and 3' restriction site.

[0191]A PCR reaction may be performed to fuse the above PCR products together into a single molecule. Products from the two PCR reactions described above may be melted and re-annealed to allow for the region of overlap from the two molecules to hybridize. For example, an equal mass of the pooled H3-3 library (approximately two uL of each pooled PCR reaction) and the H3-R fragment may be annealed at their regions of overlap. Next, amplification of annealed molecules with both the Asc-F2 primer (SEQ ID NO: 284) and the Notl-R primer (SEQ ID NO: 247) allows for the synthesis of a single contiguous molecule (see, e.g., Sambrook and Russell, Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001).

[0192]The DNA fragment synthesized by the methods above may be cloned into a pXOMA Fab or pXOMA Fab-gIII vector. Briefly, the DNA fragment is purified by using the QIAGEN® PCR purification kit and sequentially digesting the fragment with Notl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) and Ascl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) (See, Methods in Molecular Biology, vol. 178: Antibody Phage Display: Methods and Protocols Edited by: P. M. O'Brien and R. Aitken, Humana Press, "Standard Protocols for the Construction of Fab Libraries, Clark, M. A., 39-58) (see, e.g., FIG. 6). Next, the vectors may be ligated with the mutagenized insert using T4 Ligase (NEW ENGLAND BIOLABS®, Ipswich, Mass.) and transformed into TG1 cells by electroporation.

Example 3

Selection of High Affinity Binders

[0193]Phage containing a modified antibody variable domain that binds to an antigen (e.g., Ep-Cam or IL-1β) with high affinity may be selected by standard panning protocols (see, e.g., Methods in Molecular Biology, vol. 178: Antibody Phage Display: Methods and Protocols Edited by: P. M. O'Brien and R. Aitken, Humana Press, "Panning of Antibody Phage-Display Libraries", Coomber, D. W. J. pp 133-145, and "Selection of Antibodies Against Biotinylated Antigens", Chames, P. et al. p. 147-157).

[0194]In an exemplary method, library phage for the panning procedure are amplified by inoculating fifty milliliters of 2YT with library TG1 cells and grown to an OD₆₀₀ of 0.6-0.8. Helper phage VCSM13 are added to the inoculated 2YT culture at a multiplicity of infection (M.O.I.) of 10 (e.g., in 50 mL of cells with OD₆₀₀=0.6 there are 0.6×3⁸×50=9×10⁹ cells, M.O.I. of 10 is therefore 9¹⁰ helper phage, which corresponds to about 10 μl of 1¹³ stock phage). The helper phage are used to infect the TG1 cells by gently mixing the phage with the cells with no shaking for thirty minutes. The culture is then shaken for an additional thirty minutes at 180 rpm. Following infection, the culture is spun down at 2500 rpm for ten minutes. The resulting cell pellet is resuspended in fifty milliliters of 2TYAmpKan and grown overnight at 30° C. and the supernatant is removed and discarded.

[0195]Exemplary methods of panning include coating one well of a NUNC® MAXISORP plate with fifty μl of Ep-Cam or IL-1β at 0.1 μg/ml in DULBECCO'S® PBS with Calcium and Magnesium chloride (Invitrogen, Carlsbad, Calif.) and incubating the plates overnight at 4° C. The wells are then blocked with 5% milk in PBS for one hour at room temperature. Separately 0.5 ml of phage supernatant from the overnight culture described above are blocked with 300 μL of 10% milk in PBS for one hour at room temperature. Blocked phage (e.g., approximately 200 μl) are added to the blocked wells in 3% BSA-PBS and incubated at room temperature with shaking for one to two hours. After incubation, the wells are emptied and washed five times with PBST quick wash (e.g., PBS+0.05% Tween 20), then washed five times with PBST five minute wash, followed by five washes with PBS quick wash and lastly washed five times with PBS five minute wash. Phage bound to the wells are eluted by incubating with 200 μL/well of freshly prepared 100 mM TEA (prepared by adding 140 μL of 7.18 M Triethylamine stock to ten ml H₂O for 20 minutes at room temperature (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001). The eluate is transferred to a Falcon tube containing 0.5 ml 2M TRIS-HCl pH 7.4. The pH of the eluate is checked with pH paper to ensure that it is about pH 7 and adjusted if necessary.

[0196]Eluted phage from the exemplary panning method are amplified by infecting TG1 cells. In an exemplary method, TG1 cells are grown to an OD₆₀₀=0.6 (e.g., mid log phase) and ten ml of the culture is added to the phage eluate from the panning method described above. The eluted phage are used to infect the TG1 cells at 37° C. for thirty minutes without shaking and then continued for an additional thirty minutes at 37° C. with shaking at 240 rpm. After the infection, the culture is centrifuged at 2500 rpm for five minutes. Next, the supernatant is removed and the cell pellet is resuspended in 700 μL of 2YTAG. The re-suspension is plated on two 15 cm 2YTAG agar plates and incubated at 30° C. overnight. After the overnight incubation, the cells are scraped from the two plates using five to ten milliliters of 2YTAG per plate, and transferred to a fifty milliliter falcon tube where they are used to make a glycerol stock.

[0197]In an alternative exemplary method, panning may be performed with biotinylated Ep-Cam or IL-1β. Briefly, two hundred microliters of streptavidin beads (Dynal) are blocked in 5% BSA-PBS (100 μl of the blocked beads are used for the de-selection and 100 μL for the selection). Using a magnet, the beads are removed from the 5% BSA-PBS and rinsed twice in PBS. To the rinsed beads is added one milliliter of 5% BSA-PBS and the beads are incubated at room temperature for one hour with very gentle rotation. After the incubation, the beads are split into two tubes, with the supernatant removed from one tube for the de-selection. Phage solution is added to the tube with beads designated for the de-selection and resuspended. The phage-bead solution is incubated at room temperature for forty-five minutes with gentle rotation. After the incubation, the phage supernatant (de-selected phage solution) is transferred to a new tube using a magnet. Next, the de-selected phage solution is incubated at room temperature for sixty minutes with one hundred pmols of biotinylated Ep-Cam or IL-1β. The phage-biotinylated Ep-Cam or IL-1β solution is then added to a new aliquot of streptavidin beads (with the supernatant removed) and incubated at room temperature for sixty minutes. After the incubation, the beads are separated from the supernatant using a magnet. Next the beads are washed five times with one ml of 0.5% BSA-PBST by adding the wash to the tube, closing the tube and resuspending the pellet, putting back in the magnet waiting a few seconds until the beads are attached to the magnet side of the tube and removing the wash with a pipetman. Further, the beads are washed five times in 0.5% BSA-PBST for five minutes for each wash, washed five times with one milliliter of 0.5% BSA-PBS, washed five times for five minutes each wash in five milliliters of 0.5% BSA, and washed one time with PBS. Bound phage are eluted by incubating the beads with 500 μL of freshly prepared 100 mM TEA (add 140 μL of 7.18 M Triethylamine stock to 10 ml H₂O) for thirty minutes at room temperature with gentle rotation. The eluate is separated from the beads by using a magnet and transferred to a fifty milliliter falcon tube containing 250 μl of 1M TRIS pH 7.4 to neutralize the TEA and can be used for infection and/or amplification (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001). For example, log phase TG1 cells may be infected with phage eluate at 37° C. for one hour at ninety rpm. After infection of the cells, the culture is centrifuged at 2500 rpm for five minutes and the supernatant removed. Next, the cell pellet is resuspended in 700 μl of 2YTAG, plated onto two 15 cm 2YT-ampicillin-2% glucose agar plates and incubated at 30° C. overnight.

Example 4

Screening of an Affinity Matured Antibody Using the DELFIA® Competition Assay

[0198]Individual Fabs obtained from the affinity-based selection of libraries of the ING-1 antibody clone are tested for their ability to inhibit the binding of Ep-Cam to the parent chimeric ING-1 IgG antibody using a competitive screening assay (e.g., the microplate based competitive screening DELFIA® assay (PERKIN ELMER®, Waltham, Mass.)). Ninety-six well plates containing two hundred and fifty microliters of 2YT media are inoculated with glycerol stock of Fab-expressing E. coli transformed with the pXOMA-Fab vector. The culture is grown at 37° C. until cloudy (approximate OD600=0.5) and inoculated with IPTG to a final concentration of 1 mM. The cultures are grown overnight at 30° C. In addition, a Costar plate 3922 (White) is coated with 1.25 ug/mL of parental ING-1 chimeric IgG O/N at 4° C.

[0199]Periplasmic extracts (PPE) of the overnight expression constructs are prepared by spinning the overnight expression plates at 3000 rpm for fifteen minutes, discarding supernatant and adding 60 microliters of PPB buffer (periplasmic extraction buffer, 30 mM Tris-HCl pH 8.0, 20% sucrose, 1 mM EDTA) to each well. The pellets are resuspended, and 90 microliters of cold PPB diluted 1:5 with cold water are added to each well. This mixture is incubated on ice for one hour and subsequently spun down at 3000 rpm for fifteen minutes. This PPE supernatant is transferred to a new plate. The PPE is diluted into 10% PPE in PBS, 5% PPE in PBS, and 1% PPE in PBS. For the coated Costar plate, it is washed three times with PBS-tween and blocked with 350 microliters of 3% BSA in PBS for one hour.

[0200]The blocked Costar plate is washed three times with PBS and then biotinylated Ep-Cam is added to the diluted PPE to a final concentration of 3 nM. The diluted PPE and biotinylated Ep-Cam solution is then added to the coated Costar plate and incubated for one and a half hours at room temperature. The plates are washed three times with PBST and fifty microliters of 1:250 dilution of Europrium-Streptavidin in Delfia Assay Buffer (PERKIN ELMER®, Waltham, Mass.) is added. The mixture is incubated at room temperature for one hour, and the Time-Resolved Fluorescence Plate reader is setup (Gemini microplate reader, Molecular Devices), interval 200-1600 microseconds, 20 reads/well, excitation 345 nm, emission 618 nm and cutoff 590 nm. The plates are washed seven times with Delfia Wash Buffer (PERKIN ELMER®, Waltham, Mass.), followed by the addition of fifty μl of Delfia Enhancement buffer (PERKIN ELMER®, Waltham, Mass.) and incubated for five minutes. The plates are read on the Gemini plate reader. Plates with decreased signal compared with control parental antibody show greater binding by the affinity matured Fab and can be further characterized by Biacore (e.g., Biacore 2000 or A100) and other affinity measuring techniques (see, e.g., Tables 4 and 5).

[0201]Similarly, XPA23 antibody clones may be tested for their ability to inhibit the binding of IL-1β to the parent chimeric XPA23 IgG using a competitive screening assay as described above.

TABLE-US-00004 TABLE 4 Delfia Screening of 10% Periplasmic Extract 1 2 3 4 5 6 7 8 9 10 11 12 A 46.9 37.1 71.2 75.7 51.3 22.3 65.8 72.9 58.8 81.7 56.2 96.7 B 2.6 55.2 39.2 54.8 31.7 41.3 57.1 56.7 21.6 77.8 1.8 102.0 C 53.2 42.3 72.5 61.2 16.2 78.0 41.2 57.2 63.8 28.6 13.6 100.7 D 49.0 45.5 8.9 1.0 21.5 82.8 105.8 67.3 68.5 61.8 63.5 100.6 E 49.1 72.1 68.6 0.3 91.8 57.6 53.1 8.3 58.3 60.4 82.2 -0.4 F 61.7 72.1 71.8 45.6 44.6 53.1 15.3 73.2 84.7 15.1 59.0 0.1 G 58.4 26.4 1.0 59.4 62.3 19.9 -0.1 49.0 52.4 76.2 46.8 0.3 H 36.1 67.7 65.2 27.4 34.3 50.3 60.0 60.1 56.8 83.0 49.3 -0.4 Percentage of inhibition is shown in each well using the average signal from wells A12-D12 as positive control, 100% inhibition and the average signal in well E12-H12 as 0% inhibition negative control wells. Wells bolded show strong competition in the Delphia assay.

TABLE-US-00005 TABLE 5 ##STR00001## Percentage of inhibition is shown in each well using the average signal from wells A12-D12 as positive control, 100% inhibition and the average signal in well E12-H12 as 0% inhibition negative control wells. Wells bolded show strong competition in the Delphia assay. Boxed wells retain strong inhibition and are prioritized for affinity testing.

Example 5

Screening of an Affinity Matured Antibody Using Kinetic Titration Analysis

[0202]Kinetic properties of affinity matured antibodies, for example, as represented by XPA23 clones such as Y208L may be determined by kinetic titration analysis. In an exemplary method, an antigen such as IL-1β is amine coupled to a CM5 sensor chip. Each sample (e.g., from lowest to highest concentration) may be injected for 240 seconds at a flow rate of 30 μl/min at a selected temperature (e.g., 25° C.). Sample are allowed to dissociate for 30 seconds except the highest concentration which may be permitted 300 seconds to dissociate. The assay is run at 25° C.

[0203]Biaevaluation software (e.g., Biacore 2000 evaluation software) is used to calculate dissociation rates of individual samples and the relative amount of sample bound to each test surface. The data is fit to an appropriate kinetic model (e.g., the kinetic titration model). For example, XPA23 had a ka=2.5e5 and a kd=1.2e-2 KD=4.6e-8, while the modified XPA23 Y208L mutant had a ka=3.57E+05 kd=5.80E-03 KD=1.62E-08.

Example 6

ELISA Measurement for Fab Expression or Antigen Binding

[0204]Additionally or alternatively to the Biacore assay described below in Example 10, an ELISA assay may be used for the identification of modified antibody variable domains that bind its binding partner or for verifying expression of Fab domains.

[0205]In an exemplary method, ELISA plates (e.g., Nunc MAXISORP®) are coated with 1 μg/ml EpCam, 1 μg/mL EpCam for EpCam ELISA, 1 μg/mL IL-1 (Peprotech), or anti-human IgG, F(ab')₂ fragment specific antibody (Jackson Immunoresearch) in PBS at 50 μg/ml. The ELISA plates are then covered and incubated at 4° C. overnight. After the incubation, the coated ELISA plates are washed three times with PBS. The plates are then filled with 370 μl of 3% milk (e.g., Carnation, nonfat) and incubated for one hour at room temperature. Separately, 150 μl of periplasmic extract is blocked by adding 50 μl of 15% milk and incubating the extract for one hour at room temperature. The blocked plates are washed three times with PBS and 50 μL of the blocked periplasmic extract is added to each well of the antigen coated ELISA plates. The plates are incubated for two hours at room temperature and then washed four times with TBST.

[0206]Secondary antibodies are added to each ELISA plate. For the Ep-Cam or IL-1 ELISA, 50 μl of mouse anti-human c-myc antibody (9E10 Ab, Roche) at 2.5 μg/ml in 3% milk is added to each well. For the anti-Fab ELISA, 50 μl of biotin-SP-conjugated anti-human IgG F(ab')2 fragment specific antibody (Jackson lmmunoresearch) at 1:2000 dilution in 3% milk is added to each well. The plates from both ELISAs are incubated at room temperature for one hour. After the incubation, the plates are washed four times with TBST. After the washes, a tertiary antibody may be added to the plates in both ELISAs. For the Ep-Cam or IL-1 ELISA, 50 μl of goat anti-mouse IgG-HRP (Pierce) diluted 1:10,000 in 3% milk is added to each well. For the anti-Fab ELISA, 50 μl of extravidin-HRP conjugate (Sigma) at a 1:500 dilution in 3% milk is added to each well. Again the plates from both ELISAs are incubated for one hour at room temperature. After the incubation, the plates are washed four times with TBST. Next, 50 μl of the TMB substrate (Calbiochem) is added to each well and incubated until the color develops (do not incubate long enough to see the negative control turn blue). The reaction is stopped by adding 50 μl of 2N H₂SO₄ to each well and the plates are read at 450 nm.

Example 7

Methods for Off-Rate Ranking of Antibodies or Fragments Thereof

[0207]A high-throughput off-rate ranking method is used for rapid prioritization of modified antibody variable domains that bind to their binding partner by analyzing their relative off-rates (using, e.g., Biacore 2000 or A100).

[0208]In an exemplary method, modified antibody variable domains (e.g., Epcam-binding) are produced in ninety-six well plates by inoculating two hundred and fifty microliters of 2YT media with a glycerol stock of Fab-expressing E. coli transformed with a pXOMA-Fab vector comprising a modified Epcam-binding variable domain. The culture is grown at 37° C. until cloudy (e.g., approximate OD₆₀₀=0.5), inoculated with IPTG to a final concentration of 1 mM and grown overnight at 30° C.

[0209]Next, periplasmic extracts (PPE) of the overnight expression constructs are prepared by spinning the overnight expression plates at 3000 rpm for fifteen minutes, discarding the supernatant and adding 60 μl of PPB buffer to each well. The pellets are resuspended, and 90 μl of cold PPB diluted 1:5 with cold water is added to each well. This mixture is incubated on ice for one hour and subsequently spun down at 3000 rpm for fifteen minutes. The supernatant is transferred to a new plate and the periplasmic extracts are used for the Biacore (e.g., Biacore 2000 or A100) determination.

[0210]Epcam from the periplasmic extracts is amine coupled (e.g., 10 μpg/mL Epcam in pH 4.5 acetate, seven minute injection at 5 μl/minute) to a CM5 sensor chip and periplasmic extracts containing the antibody fragments are injected over the sensor, resulting in binding of the Fab to the immobilized Epcam. Non specific binding of the antibody fragment to the sensor surface is corrected by subtracting the interaction of the antibody fragment with a blank flow cell (e.g., having no immobilized Epcam) from the interaction of the antibody fragment with the Epcam immobilized flow cell. The instrument settings are: a flow rate of 20 microliters/minute, an injection time of three minutes, a dissociation time of five minutes and an instrument temperature set to 25° C. Biaevaluation software is used to calculate dissociation rates of individual samples and the relative amount of sample bound to each test surface. Samples are then ranked according to their dissociation rates. Sensograms depicting the off-rates for heavy chains (FIG. 15) and light chains (FIG. 16) are shown. The off rates for the improved clones are tabulated for the heavy chain (FIG. 11) and the light chain (FIG. 12).

[0211]Likewise, modified XPA23 variable domains (e.g., IL-1β-binding) may be ranked according to their dissociation rates using the high-throughput off-rate ranking method described above. The instrument settings are: a flow rate of 30 microliters/minute, an injection time of three minutes, a dissociation time of ten minutes and an instrument temperature set to 25° C. The off rates for the improved clones are tabulated for the heavy chain (FIG. 13) and the light chain (FIG. 14).

[0212]The modified antibody variable domains of the present disclosure may have a k_off that is greater than (see, e.g., FIG. 20), less than (see, e.g., FIG. 18) or equal to (see, e.g., FIG. 19) than an unmodified antibody variable domain.

Example 8

Reformatting of Candidate Clones to IgG

[0213]Two of the improved off-rate clones from the k_off analysis were reformatted into IgG₁ format by PCR amplification of the heavy and light chain variable domains and cloning the PCR amplified regions into a mammalian expression vector containing the Fc and the light chain constant domain respectively. The heavy chain is cloned into a mammalian expression vector containing a CMV promoter using Bsml and Nhel sites for the 5' and 3' ends respectively and is cloned in frame with the heavy chain secretion signal on the 5' end and the constant CH1,CH2, and CH3 portions of the IgG molecule on the 3' end. The amplification sequences are as follows: (ING-HC-IgGF 5'-ATATATTGCATTCCCAGATCCAGTTGGTGCAGTC-3'), ING-HC-IgGR (5'-ATATATGCTAGCTGAGCTGACGGTGACCGAGGTTCC-3'). The light chain is cloned into a similarly constructed expression vector utilizing a blunt 5' cloning site and the BsiWI site on the 3' end and is cloned in frame with the light chain secretion signal on the 5' end and the light chain constant region on the 3' end. The PCR amplification primer sequences are as follows: (ING-LC-IgGF 5'-CAAATTGTGATGACGCAGGC-3') and (ING-LC-IgGR 5'-ATATATCGTACGTTTCATCTCTAGTTTGGTGCC-3'). The PCRs are performed under standard conditions: see, e.g., Sambrook and Russell, Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001. Improved off-rate clones reformatted into IgG, vectors are transiently co-transfected in a 2:1 light chain to heavy chain DNA ratio into HEK 292 cells using Lipofectamine 2000 (Invitrogen) using the manufacturer's guidelines. Secreted IgGs secreted from HEK 292 cells are purified using protein A SEPHAROSE® (GE-AMERSHAM® Piscataway, N.J.) using the manufacturer's guidelines and tested by BIACORE® (e.g., Biacore 2000 or A100) for affinity (see, e.g., FIGS. 11 and 15) and Example 8.

Example 9

Expression and Testing of Modified Antibody Variable Domains with a Combination of Amino Acid Changes

[0214]Modified antibody variable domains with improved off-rates and affinities as compared to a parent variable region may be identified by employing the DELFIA® competition assay and/or BIACORE® (e.g., Biacore 2000 or A100) off-rate ranking. Clones with improved k_off are sequenced and aligned by both their light and heavy chain. Identified amino acid changes in the light and heavy chain that increase affinity can be combined in one modified antibody variable domain for potential additive and synergistic combinations. Modifications for combination may utilize the residues that have improved off-rates greater than or equal to 4.9 fold compared with the parental antibodies (see, e.g., FIGS. 11, 12). For any given amino acid position, the change that leads to the greatest improvement is chosen for study. This compilation is described in Table 6, and will lead to 21 combinations of heavy and light chains (e.g., 7 heavy chains combined in all variations with three light chains).

TABLE-US-00006 TABLE 6 Heavy and Light Chain CDR1, CDR2 and/or CDR3 Combinations CDR1 CDR2 CDR3 Heavy Chain Combinations G33F wt wt wt T53I wt wt wt G100R G33F T53I wt wt T53I G100R G33F wt G100R G33F T53I G100R Light Chain Combinations wt Q55R wt wt wt E98T wt Q55R E98T

[0215]Alternatively, the initial modifications for combination may utilize the residues that have improved off-rates greater than or equal to approximately 2.5-fold compared with the parental antibodies (see, e.g., FIGS. 13, 14). For any given amino acid change, the change that leads to the greatest improvement is chosen for study. The amino acids with greater than or equal to approximately 2.5 fold improved k_off are compiled in Table 7. There are two amino acids in CDR1 (position 28), two amino acids in position 100, three amino acids in position 101, and five amino acids in position 102. In all, there are 60 (2×2×3×5=60) combinations.

TABLE-US-00007 TABLE 7 Heavy Chain CDR1 and CDR3 Combinations CDR1 CDR3 28T (wt) 100G (wt) 28I 100R 101S (wt) 101I 101G 102A (wt) 102Y 102F 102W 102G

[0216]A PCR based strategy may be used to create a modified antibody light chain containing more than one amino acid change (see, e.g., FIG. 7). In an exemplary method, PCR may be used to amplify three segments of the V_k gene, two of which may be engineered to contain an amino acid change. For example, to create a light chain containing the mutations Q55R and E98T, PCR product 1 may be synthesized using the HindIII-F (SEQ ID NO: 814) and L2R primer (SEQ ID NO: 74), PCR product 2 may be synthesized using L2-Q55R primer (SEQ ID NO: 808) and the L3R primer (SEQ ID NO: 110) and PCR product 3 may be synthesized using L3-E98T primer (SEQ ID NO: 807)and the Ascl-R primer (SEQ ID NO: 812). The PCR products are then melted and re-annealed such that their regions of overlap hybridize. Subsequently, all three PCR products may be joined into one molecule by PCR amplification using the forward primer from PCR product 1 (HindIII-For) (SEQ ID NO: 814) and the reverse primer from PCR product 3 (Ascl-R) (SEQ ID NO: 812). In an exemplary method to create a heavy chain containing the mutations outlined above and described in FIG. 7, product 1 may be synthesized using the Ascl-F (SEQ ID NO: 813) and H1R primer (SEQ ID NO: 146), PCR product 2 may be synthesized using H1-28TI primer and the H3R primer (SEQ ID NO: 247) and PCR product 3 may be synthesized using each H3 combination primer (6 primers, 6 r×ns) and the Notl-R primer (SEQ ID NO: 285). The PCR products are then melted and re-annealed such that their regions of overlap hybridize. Subsequently, all three PCR products may be joined into one molecule by PCR amplification using the forward primer from PCR product 1 (Ascl-F) (SEQ ID NO: 813) and the reverse primer from PCR product 3 (Notl-R) (SEQ ID NO: 285).

[0217]In an exemplary method, a 50 μL PCR reaction for the production of PCR product 1, 2 and 3 may be performed with 25 pmol of each of the forward and reverse primers, 10 ng of template DNA, 5 μL PFU buffer, 2.5 μL of 10 μM dNTPs, 1 μL PFU and water to 50 μL. The PCR reaction is heated to 94° C. for two minutes, followed by 25 cycles of 30 seconds at 94° C., 30 seconds at 54° C., and one minute at 72° C. After the 25 cycles, a final 72° C. incubation may be performed for five minutes.

[0218]An equal mass of the three PCR products may be combined in a PCR reaction to produce a modified variable domain with several amino acid changes which enhance affinity. Briefly, the PCR may be conducted by adding approximately 2 μL of each pooled PCR reaction to 5 μL PFU buffer, 25 pmol of both HindIII-f primer (SEQ ID NO: 814)and Ascl-R primers (SEQ ID NO: 812), 2.5 μL of 10 μM dNTPs, 1 μL PFU polymerase and water to 100 μL. Next, the PCR reaction is heated to 94° C. for two minutes, followed by twenty-five cycles of thirty seconds at 94° C., 30 seconds at 54° C., and finally one minute at 72° C. After the twenty cycles, a final 72° C. incubation is performed for five minutes.

[0219]The resulting DNA fragment may be purified (e.g., using the QIAGEN® PCR purification kit (Valencia, Calif.)) and sequentially digested with HindIII (NEB) and then Ascl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) such that it may be cloned into the pXOMA Fab or pXOMA Fab-gIII vector.

[0220]For the heavy chain modifications, a similar PCR based strategy may be used to create a modified antibody heavy chain containing more than one amino acid change (see, e.g., FIG. 8). In an exemplary method, PCR may be used to amplify four segments of the V_H gene, three of which may be engineered to contain the G33F, T53I and G100R amino acid changes. For example, PCR product 1 may be synthesized using the Ascl-F (SEQ ID NO: 813) and H1R primers (SEQ ID NO: 146), PCR product 2 may be synthesized using the H1-G33F primer (SEQ ID NO: 809) and H2R primer (SEQ ID NO: 182), PCR product 3 may be synthesized using H2-T3I primer (SEQ ID NO: 810) and H3R primer (SEQ ID NO: 247) and PCR product 4 may be synthesized using H3-G100R primer (SEQ ID NO: 811) and the Notl-R primer (SEQ ID NO: 285). The PCR products are then melted and re-annealed such that their regions of overlap hybridize. All four PCR products may then be joined into one molecule by PCR amplification using the forward primer from PCR product 1 (Ascl-F) (SEQ ID NO: 813) and the reverse primer from PCR product 3 (Notl-R) (SEQ ID NO: 285).

[0221]In an exemplary method, a 50 μL PCR reaction for the production of PCR products 1, 2, 3 and 4 may be performed with 25 pmol each of the forward and reverse primers, 10 ng of template DNA, 5 μL PFU buffer, 2.5 μL of 10 μM dNTPs, 1 μL PFU and water to 50 μL. The PCR reaction is heated to 94° C. for 2 minutes, followed by 25 cycles of 30 sec at 94° C., 30 seconds at 54° C., and one minute at 72° C. After the 25 cycles, a final 72° C. incubation may be performed for five minutes.

[0222]An equal mass of the four PCR products may be combined in a PCR reaction to produce a modified variable domain with several amino acid changes which enhance affinity. Briefly, the PCR may be conducted by adding approximately 2 μL of each pooled PCR reaction to 5 μL PFU buffer, 25 pmol of both Ascl-F primer (SEQ ID NO: 813) and Notl-R primer (SEQ ID NO: 285), 2.5 μL of 10 μM dNTPs, 1 μL PFU polymerase and water to 100 μL. Next, the PCR reaction is heated to 94° C. for two minutes, followed by twenty-five cycles of thirty seconds at 94° C., 30 seconds at 54° C., and finally one minute at 72° C. After the twenty cycles, a final 72° C. incubation is performed for five minutes.

[0223]The heavy chain PCR fragments and the vector will be digested with Ascl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) and Notl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) such that it may be cloned into the pXOMA Fab or pXOMA Fab-gIII vector.

Example 10

Biacore Measurement of IgG Affinity

[0224]IgGs that bind Epcam in Example 7 are tested by BIACORE® for affinity (see, e.g., FIG. 15). For example, kinetic analysis of anti-Epcam mAb's are conducted on a Biacore 2000®.

[0225]In an exemplary method, the ING1 antibody is diluted to 0.5 μg/mL in HBS-EP running buffer and injected for two minutes at 5 μl/ minute over a high density protein A/G surface. Next, six serial 3 fold dilutions of Epcam are prepared in running buffer and injected in triplicate in random order over the high density protein A/G surface with buffer injections evenly distributed throughout the run. The sample injections are then double referenced against the blank flow cells and buffer injections to correct for any bulk shift or non-specific binding. Data are then analyzed with the Biaevaluation software from Biacore and sensorgrams are fit utilizing the 1:1 langmuir model (see, e.g., FIG. 15).

Example 11

Construction of Arrays of Modified Antibody Variable Domains

[0226]Arrays of modified antibody variable domains (e.g., modified ING-1 variable domains) with amino acids changes at desired positions (e.g., contacting (C) residues) may be generated and tested for enhanced binding affinity compared to the parent variable domain (e.g., ING-1). Modified variable domains used in the array may be obtained directly from a library of modified variable domains as described in Example 2 or may first be screened for those modified variable domains that exhibit enhanced binding as compared to the parent variable domain as described in Examples 3, 4 and 5.

[0227]In an exemplary method, each contacting (C) residue in the heavy and light chain variable region of ING-1 is separately changed (e.g., by PCR mutagenesis) with alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine to generate modified ING-1 variable domains. CDNAs encoding the modified ING-1 variable domains are then inserted into a pXOMA vector and used to transform electrocompetent TG1 cells. The clones are plated on 2YT-Amp₁₀₀/2% Glucose plates (Teknova) and the plates filled with 250 μl of 2YT-Amp₁₀₀/well (Teknova). Each well is inoculated with a single colony comprising a single amino acid change at a contacting (C) residue. The colonies are grown by incubating the plates at 37° C. for two to four hours with shaking at 450 rpm. After the incubation, the plates are duplicated to sequencing plates by filling new deep-well culture plates (Thomson) with one milliliter of 2YT-Amp₁₀₀Gluc₂%/well from the grown cultures. The Genetix 96-pin replicator is used to transfer cells from the master plate to the new sequencing plates. The sequencing plates are grown overnight at 37° C. with shaking at 450 rpm. After the incubation, the sequencing plate is spun down at 5000 rpm for ten minutes and the supernatant is discarded. Samples from the plate are sequenced (e.g., samples may be submitted for automated miniprep and automated sequencing (Elim biopharmaceuticals). After the incubation, Master Plates are made by adding glycerol to a final concentration of 15% to the wells on the glycerol plate and storing the plates at -80° C. The unique clones and their well position in the master plate are identified after sequencing results are returned.

[0228]Eighteen different clones, each containing an amino acid change at a contacting (C) residue in ING-1, are identified (typically 96 sequenced clones yield all eighteen clones). Unique clones from the master plates are rearrayed to a new 96-well master plate containing 2YT-Amp₁₀₀ by transferring ten microliters of glycerol stock from the master plate to the rearrayed master plate. Alternatively, automation, such as the QPIX II is used to transfer the glycerol stock containing the unique clones to the new master plate. The new rearrayed glycerol master plates are replicated into new expression plates to perform Biacore (e.g., Biacore A100) analysis (see, e.g., Table 8 and Table 9). Arrays may also be constructed for XPA 23 modified antibodies (see, e.g., Table 10 and 11).

TABLE-US-00008 TABLE 8 Biacore Analysis of Modified Light Chain Variable Regions ¹, 2 3 NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1 K 27 1.26 -1.00 1.26 ? 1.06 nd -1.00 1.62 1.52 -1.00 S 28 1.63 1.02 2.78 2.32 1.90 2.02 nd 2.38 1.99 -1.00 L 29 -1.00 -1.00 -1.00 nd nd -1.00 -1.00 -1.00 -1.00 1.85 L 30 1.47 -1.00 -1.00 -1.00 1.45 1.53 -1.00 1.56 1.60 1.45 H 31 0.71 0.68 0.06 0.05 0.95 2.16 1.66 nd 0.57 0.50 S 32 0.94 nd 1.79 1.32 1.13 1.37 1.27 1.64 1.10 -1.00 N 33 0.49 0.65 0.71 0.70 0.73 0.80 1.04 0.93 0.73 1.38 I 35 0.19 0.16 0.92 0.61 0.59 0.51 0.34 0.66 0.41 0.50 T 36 0.05 1.60 -1.00 1.15 0.79 nd 1.30 nd 1.04 0.74 Y 37 nd 0.01 nd 0.02 4.07 0.95 0.85 0.63 0.02 0.06 CDR2 Y 54 0.03 0.05 -1.00 3.62 -1.00 0.92 0.96 0.94 1.23 -1.00 Q 55 0.05 0.05 5.31 0.46 3.82 nd 4.11 0.86 0.95 0.36 M 56 1.36 0.71 0.92 0.98 1.32 1.21 1.29 1.40 1.12 0.99 S 57 0.95 0.93 1.17 1.54 1.01 -1.00 2.34 0.96 -1.00 1.17 N 58 nd 0.97 1.77 1.40 1.16 1.43 1.99 1.03 1.55 0.95 CDR3 L 97 -1.00 0.75 -1.00 0.61 0.42 0.98 1.59 0.93 0.91 0.48 E 98 1.62 0.98 3.08 2.22 1.23 1.23 1.10 1.43 1.41 -1.00 L 99 0.02 0.01 0.04 0.02 0.05 1.00 0.89 0.43 0.02 2.00 P 100 0.02 0.06 0.05 0.03 0.05 1.94 1.51 1.65 0.04 0.05 R 101 -1.00 -1.00 0.93 0.04 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 NP Polar Aliphatic Small NP S T V I L A C G P M CDR1 K 27 -1.00 -1.00 nd 1.15 1.39 1.10 nd 1.11 1.29 nd S 28 1.08 1.19 2.15 2.35 2.60 1.53 nd nd 2.42 nd L 29 2.03 nd nd -1.00 ? 1.97 nd 1.53 -1.00 nd L 30 1.43 1.89 1.69 0.96 0.97 1.27 nd 1.64 0.82 nd H 31 0.82 1.94 1.17 1.14 0.59 1.26 nd 0.73 1.19 nd S 32 ? nd 0.93 1.17 1.12 1.35 nd 1.39 0.83 nd N 33 0.48 0.60 -1.00 nd nd 0.76 nd 0.72 0.67 nd I 35 0.55 0.87 1.08 nd 0.60 0.46 nd 0.39 0.70 nd T 36 1.10 0.94 0.98 0.76 1.64 1.02 nd 1.09 0.67 nd Y 37 0.09 -1.00 -1.00 0.90 nd 0.04 nd 1.30 nd nd CDR2 Y 54 0.90 0.61 1.32 -1.00 3.44 0.08 nd 1.85 0.86 nd Q 55 0.56 0.66 1.53 1.42 0.71 0.64 nd 0.70 0.95 nd M 56 1.05 0.80 -1.00 1.37 0.74 0.86 nd 0.80 1.38 nd S 57 1.00 0.86 0.89 0.98 1.38 1.15 nd -1.00 nd nd N 58 1.65 1.42 2.84 2.51 1.47 1.79 nd 1.87 3.47 nd CDR3 L 97 0.79 0.54 1.44 2.62 0.93 0.50 nd -1.00 0.95 nd E 98 -1.00 4.90 nd nd 2.82 1.35 nd 1.63 -1.00 nd L 99 0.04 0.09 1.04 2.07 0.93 1.43 nd 0.02 0.01 nd P 100 0.06 0.08 0.14 0.14 0.03 0.05 nd 1.62 1.01 nd R 101 -1.00 -1.00 -1.00 -1.00 1.33 -1.00 nd -1.00 -1.00 nd ¹ A value of -1 indicates no binding ² Bolded values indicate the highest affinity o affinity (as measured by how many "fold" differences in affinity. The mutant is in comparison to original, e.g., 2.0 as twice as strong and 0.5 as half as strong) obtained for an amino acid change at the position ³ nd indicates that binding affinity was not determined

TABLE-US-00009 TABLE 9 Biacore Analysis of Modified Heavy Chain Variable Regions ¹, 2, 3 NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1 T 28 0.98 1.23 1.23 1.77 1.15 1.86 1.08 1.28 -1.00 0.69 T 30 0.63 0.73 1.39 0.94 nd 2.00 1.26 nd nd 0.73 K 31 0.66 0.54 0.76 0.96 0.78 1.00 1.02 nd nd -1.00 Y 32 nd 0.08 0.43 0.08 0.60 0.84 nd 1.05 0.10 nd G 33 -1.00 -1.00 0.03 -1.00 0.02 6.16 -1.00 7.19 0.06 -1.00 CDR2 W 50 3.27 -1 0.10 0.04 0.02 0.04 0.97 0.09 0.01 0.03 N 52 0.02 -1 -1 -1 0.02 -1 -1 -1 -1 0.98 T 53 -1 -1 -1 1.79 -1 -1 -1 -1 -1 -1 Y 54 0.05 0.07 3.72 3.62 1.00 0.92 0.96 0.65 0.66 2.11 T 55 0.03 -1 0.14 0.45 0.05 0.03 0.10 0.03 0.03 0.17 E 56 0.81 0.95 1.34 1.27 1.74 1.04 1.17 0.78 1.23 1.01 E 57 1.17 1.07 1.71 nd 1.16 1.37 1.39 1.06 -1.00 1.57 P 58 0.54 0.44 nd 1.14 nd 0.99 1.11 0.98 1.11 0.90 T 59 0.87 0.51 1.22 1.43 0.40 nd 2.24 0.43 -1.00 0.96 CDR3 G 100 -1 -1 7.51 1.59 -1 -1 -1 -1 -1.00 1.55 S 101 0.21 0.76 nd 2.20 1.35 1.79 1.22 1.16 2.18 0.97 A 102 0.28 0.51 2.18 1.48 2.40 3.01 3.13 2.97 1.01 0.94 D 104 nd 0.14 -1 -1 -1 -1 -1 -1 -1 0.73 Y 105 -1 -1 0.66 -1 0.94 nd 0.84 0.91 0.87 nd NP Polar Aliphatic Small NP S T V I L A C G P M CDR1 T 28 1.07 nd 2.08 2.45 nd 1.56 nd 0.92 2.16 nd T 30 0.91 0.93 1.30 nd 1.26 0.89 nd -1.00 0.93 nd K 31 0.49 nd 1.41 1.17 0.60 0.39 nd 1.02 nd nd Y 32 0.01 nd 0.11 0.03 0.05 0.01 nd 0.02 0.01 nd G 33 nd 0.01 0.04 0.55 2.27 0.06 nd nd 6.31 nd CDR2 W 50 0.02 0.03 0.02 0.07 0.04 0.03 nd 0.03 -1 nd N 52 -1 -1 -1 -1 -1 -1 nd -1 -1 nd T 53 0.17 1.19 2.44 11.40 nd 9.03 nd -1 -1 nd Y 54 0.49 0.36 1.32 0.28 1.80 0.47 nd 3.72 0.86 nd T 55 0.42 nd 0.28 nd 0.95 nd nd 0.02 nd nd E 56 1.46 1.21 0.86 0.85 0.64 1.67 nd 1.37 0.01 nd E 57 1.41 1.44 nd 1.34 -1 1.65 nd 1.45 -1.00 nd P 58 1.07 1.00 1.01 nd 0.64 1.03 nd 1.31 1.06 nd T 59 nd 0.99 1.03 0.76 0.95 nd nd 0.35 -1.00 nd CDR3 G 100 1.68 0.61 2.17 nd 0.65 1.99 nd nd -1 nd S 101 nd 0.84 1.92 3.53 -1 1.15 nd 3.31 nd nd A 102 0.94 0.68 1.20 0.79 -1 nd nd 3.58 0.87 nd D 104 1.87 -1 -1 nd -1 -1 nd 0.41 -1 nd Y 105 0.09 0.12 0.18 0.23 0.22 0.08 nd -1.00 -1 nd ¹ A value of -1 indicates no binding ² Bolded values indicate the highest affinity o affinity (as measured by how many "fold" differences in affinity. The mutant is in comparison to original, e.g., 2.0 as twice as strong and 0.5 as half as strong) obtained for an amino acid change at the position ³ nd indicates that binding affinity was not determined

TABLE-US-00010 TABLE 10 Biacore Analysis of Modified C5A (XPA23) Light Chain Variable Regions ¹, 2, 3 NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1 Q27 1.07 1.08 0.89 -1.00 1.09 nd -1.00 2.96 1.06 -1.00 D28 1.00 0.74 0.94 0.82 1.23 1.45 3.81 1.43 nd 1.00 N30 0.81 0.64 0.74 0.61 1.00 1.40 1.06 1.59 0.60 1.08 R31 11.11 -1.00 1.06 1.18 -1.00 -1.00 0.43 0.92 0.27 0.38 W32 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 0.99 -1.00 -1.00 -1.00 CDR2 H49 0.11 -1.00 0.21 0.10 1.10 0.52 0.21 0.50 -1.00 0.31 S50 -1.00 0.02 -1.00 0.10 0.05 0.05 0.02 -1.00 0.21 0.25 A51 0.13 0.29 0.18 nd 0.61 0.45 0.24 -1.00 0.24 0.30 T52 0.72 0.61 3.37 3.23 0.91 1.01 0.87 1.05 0.83 0.83 S53 -1.00 1.13 3.29 4.07 nd 1.23 1.11 1.24 1.09 1.23 CDR3 A91 1.08 0.10 -1.00 nd 1.12 -1.00 -1.00 -1.00 -1.00 0.10 D92 0.83 0.99 -1.00 0.23 0.63 -1.00 0.12 0.26 0.56 0.67 S93 4.59 3.71 0.91 0.86 1.08 1.45 5.49 1.32 1.54 3.76 F94 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 1.14 -1.00 -1.00 P95 1.32 0.88 -1.00 -1.00 -1.00 -1.00 -1.00 0.98 -1.00 -1.00 L96 -1.00 -1.00 -1.00 -1.00 0.07 -1.00 4.49 0.28 0.21 0.06 NP Polar Aliphatic Small NP S T V I L A C G P M CDR1 Q27 3.30 0.64 -1.00 0.82 0.83 1.47 nd -1.00 -1.00 nd D28 5.25 1.19 0.85 -1.00 9.64 0.88 nd 1.21 1.17 nd N30 0.72 0.97 0.89 0.65 0.68 0.61 nd 0.71 0.69 nd R31 0.44 0.24 0.44 0.68 0.51 8.48 nd 0.92 -1.00 nd W32 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 nd -1.00 -1.00 nd CDR2 H49 0.14 -1.00 -1.00 -1.00 0.07 0.23 nd -1.00 0.86 nd S50 1.04 0.87 0.21 0.25 0.49 0.13 nd 0.14 -1.00 nd A51 0.95 0.76 0.31 -1.00 0.16 1.09 nd 3.84 0.13 nd T52 0.78 1.02 0.82 0.80 0.78 0.66 nd -1.00 1.08 nd S53 1.08 0.95 1.42 1.42 0.99 1.07 nd 1.03 4.92 nd CDR3 A91 0.90 0.67 0.51 -1.00 0.39 -1.00 nd -1.00 -1.00 nd D92 6.59 0.34 -1.00 0.33 0.14 0.40 nd 0.26 -1.00 nd S93 1.20 1.47 3.81 1.35 1.08 1.16 nd 0.75 -1.00 nd F94 -1.00 -1.00 0.20 0.88 0.55 -1.00 nd -1.00 0.17 nd P95 4.05 -1.00 -1.00 -1.00 -1.00 3.83 nd -1.00 -1.00 nd L96 0.23 0.06 nd 0.67 1.16 0.26 nd -1.00 -1.00 nd ¹ A value of -1 indicates no binding ² Bolded values indicate the highest affinity (as measured by how many "fold" differences in affinity. The mutant is in comparison to original, e.g., 2.0 as twice as strong and 0.5 as half as strong) obtained for an amino acid change at the position ³ nd indicates that binding affinity was not determined

TABLE-US-00011 TABLE 11 Biacore Analysis of Modified C5A (XPA23) Heavy Chain Variable Regions ¹, 2, 3 NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1 T28 nd -1.00 -1.00 -1.00 -1.00 nd -1.00 nd -1.00 nd S30 -1.00 -1.00 0.10 -1.00 -1.00 -1.00 0.77 -1.00 -1.00 0.15 K31 0.04 -1.00 0.90 1.30 1.11 0.04 0.84 0.05 -1.00 -1.00 Y32 0.68 0.12 nd nd 0.62 nd nd 0.75 -1.00 0.04 F33 0.92 0.86 -1.00 0.85 -1.00 0.77 0.79 -1.00 0.78 0.97 F35 0.06 -1.00 -1.00 0.68 -1.00 0.85 -1.00 -1.00 -1.00 0.86 CDR2 V50 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 0.03 I51 0.07 0.73 0.10 0.11 0.08 1.75 -1.00 0.90 0.83 0.68 S52 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 P53 -1.00 0.03 nd 0.04 -1.00 0.03 0.05 0.05 0.03 0.06 S54 0.12 0.03 1.43 1.53 nd 0.99 1.07 0.87 1.01 0.08 G55 -1.00 0.11 0.95 0.11 nd 0.08 nd nd 0.90 0.10 G56 0.05 1.21 2.11 2.05 0.82 1.27 1.51 1.41 1.31 1.06 M57 -1.00 -1.00 0.06 0.04 0.08 0.86 nd 1.03 0.02 0.03 T58 0.12 -1.00 1.14 -1.00 0.98 1.01 0.93 1.04 -1.00 0.90 R59 -1.00 -1.00 -1.00 0.94 -1.00 -1.00 0.09 0.92 0.86 0.86 CDR3 V99 nd -1.00 nd -1.00 nd nd nd 0.03 -1.00 -1.00 G100 -1.00 nd -1.00 -1.00 0.06 0.09 0.05 -1.00 -1.00 nd Y101 -1.00 0.03 0.85 nd 0.04 1.00 0.81 0.85 0.06 0.06 G102 nd -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 nd -1.00 1.17 G103 -1.00 -1.00 -1.00 -1.00 nd nd -1.00 -1.00 -1.00 nd N104 -1.00 -1.00 -1.00 -1.00 0.07 -1.00 -1.00 -1.00 0.03 1.02 S105 nd 1.17 -1.00 nd 0.05 -1.00 -1.00 1.25 0.98 0.85 D106 0.98 0.04 0.06 0.04 0.02 -1.00 -1.00 -1.00 0.02 0.07 Y107 0.85 0.90 0.85 0.82 -1.00 -1.00 0.89 0.90 0.87 -1.00 NP Polar Aliphatic Small NP S T V I L A C G P M CDR1 T28 nd -1.00 nd nd nd nd nd -1.00 nd nd S30 0.85 0.06 -1.00 0.08 0.91 0.77 nd 0.13 -1.00 nd K31 -1.00 -1.00 0.81 0.74 1.08 nd nd nd -1.00 nd Y32 1.33 0.80 1.07 nd 0.75 -1.00 nd 1.00 0.68 nd F33 0.73 0.78 0.76 0.75 0.88 0.75 nd 0.76 0.91 nd F35 0.09 0.74 0.89 0.85 0.78 0.07 nd 0.04 0.06 nd CDR2 V50 -1.00 0.03 0.19 0.10 0.09 0.09 nd 0.03 -1.00 nd I51 0.69 0.86 0.99 0.95 0.79 1.04 nd 0.94 -1.00 nd S52 nd 0.04 0.04 -1.00 -1.00 0.05 nd 0.03 0.05 nd P53 0.03 nd 1.10 0.08 0.04 0.05 nd 0.02 0.94 nd S54 nd 0.14 1.00 0.86 0.91 1.00 nd 1.43 0.06 nd G55 1.02 nd nd 0.86 0.08 nd nd 0.99 nd nd G56 nd 1.11 nd 1.71 1.41 1.21 nd nd 0.85 nd M57 0.04 0.06 0.03 0.03 0.06 0.03 nd nd -1.00 nd T58 0.82 1.04 0.99 0.95 0.95 0.14 nd 0.95 1.81 nd R59 0.13 0.76 -1.00 -1.00 0.08 -1.00 nd 1.04 0.07 nd CDR3 V99 -1.00 0.10 nd nd -1.00 0.03 nd nd -1.00 nd G100 2.80 0.12 nd nd -1.00 nd nd 1.02 -1.00 nd Y101 nd 0.06 0.09 0.83 1.91 0.04 nd -1.00 0.04 nd G102 nd nd -1.00 nd -1.00 -1.00 nd nd -1.00 nd G103 nd nd 2.56 nd nd 0.03 nd 0.93 nd nd N104 1.02 0.95 1.27 nd 1.19 1.61 nd 1.21 nd nd S105 1.09 0.11 0.08 0.07 -1.00 0.84 nd 0.12 1.41 nd D106 0.03 -1.00 -1.00 -1.00 -1.00 -1.00 nd 0.03 -1.00 nd Y107 0.89 0.83 0.93 0.96 0.86 0.83 nd 0.10 0.04 nd ¹ A value of -1 indicates no binding ² Bolded values indicate the highest affinity o affinity (as measured by how many "fold" differences in affinity. The mutant is in comparison to original, e.g., 2.0 as twice as strong and 0.5 as half as strong) obtained for an amino acid change at the position ³ nd indicates that binding affinity was not determined

Example 12

Construction of Arrays of Modified Antibody Variable Domains

[0229]Arrays of modified antibody variable domains (e.g., modified ING-1 variable domains) with amino acids changes at desired positions (e.g., contacting (C) residues) may be generated and tested for enhanced binding affinity compared to the parent variable domain (e.g., ING-1). Modified variable domains used in the array may be obtained directly from a library of modified variable domains as described in Example 2 or may first be screened for those modified variable domains that exhibit enhanced binding as compared to the parent variable domain as described in Examples 3, 4 and 5.

[0230]In an exemplary method, each contacting (C) residue in the heavy and light chain variable region of ING-1 is separately changed (e.g., by PCR mutagenesis) with alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine to generate modified ING-1 variable domains. cDNAs encoding the modified ING-1 variable domains are then inserted into a pXOMA vector and used to transform electrocompetent TG1 cells. The clones are plated on 2YT-Amp₁₀₀/2% Glucose plates (Teknova) and the plates filled with 250 μl of 2YT-Amp₁₀₀/well (Teknova). Each well is inoculated with a single colony comprising a single amino acid change at a contacting (C) residue. The colonies are grown by incubating the plates at 37° C. for two to four hours with shaking at 450 rpm. After the incubation, the plates are duplicated to expression plates by filling new plates (Costar) with two hundred and fifty microliters of 2YT-Amp100 media (Teknova). The Genetix 96-pin replicator is used to transfer cells from the Master plate to the new expression plates. The culture is grown at 37° C. until cloudy (e.g., approximate OD₆₀₀=0.5), inoculated with IPTG to a final concentration of 1 mM and grown overnight at 30° C.

[0231]Next, periplasmic extracts (PPE) of the overnight expression constructs are prepared by spinning the overnight expression plates at 3000 rpm for fifteen minutes, discarding the supernatant and adding 60 μl of PPB buffer to each well. The pellets are resuspended, and 90 μl of cold PPB diluted 1:5 with cold water is added to each well. This mixture is incubated on ice for one hour and subsequently spun down at 3000 rpm for fifteen minutes. The supernatant is transferred to a new plate and the periplasmic extracts are used for the Biacore (e.g., Biacore A100) determination.

[0232]After Biacore determination, wells that contain clones with improved off rates are sequenced and further characterized (e.g. IgG reformatting and affinity determination).

Example 13

Affinity Optimization of an Antibody Variable Domain by Targeted Mutagenesis of Selected Amino Acid Residues

[0233]Affinity optimized antibodies or fragments thereof may be obtained by mutation of one or more selected amino acid residues in a parent antibody or binding fragment thereof with other amino acid residues (e.g., alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine). Methods for optimization of an antibody variable domain may comprise the stages as set forth below.

A. Selection of Amino Acid Residues for Mutation

[0234]Amino acid residues at one or more positions in a parent antibody or binding fragment thereof are selected for mutagenesis. Such methods may include, for example, identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A, 3B, 3C and/or 3D. One or more amino acid residues identified as C, P, S and/or I residues may be selected for mutation.

B. Design of Primers for Mutagenesis

[0235]Primers are designed to mutagenize a parent nucleic acid sequence that codes for an antibody or binding fragment thereof.

[0236]For a PCR-based mutagenesis method, a primer may be designed such that the forward primer sequence flanks both sides (e.g., 20 base pairs) of the position to be mutated. Additionally, it is preferred that the primer be 70 bases or less in length. A representative CDR comprising amino acid residues 1-8 is shown below.

TABLE-US-00012 aa# 1 2 3 4 5 6 7 8 G F T F S K Y F 5'-G TCTTTCTTGC GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT (SEQ ID NO: 964) GGGTTCGCCAAGC-3' 3'-C AGAAAGAACG CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA (SEQ ID NO: 965) CCCAAGCGGTTCG-5'

[0237]If the CDR is too long to incorporate all the desired mutations and remain under 70 nucleotides, the mutagenesis region may be broken up into two regions. An example of this process is shown below, where the 8 amino acid CDR as shown above is broken into two 4 amino acid regions (region 1 and region 2, respectively).

Region 1:

TABLE-US-00013 [0238]aa# 1 2 3 4 G F T F 5'-G TCTTTCTTGC GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT GGGTTC-3' (SEQ ID NO: 966) 3'-C AGAAAGAACG CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA CCCAAG-5' (SEQ ID NO: 967)

Region 2:

TABLE-US-00014 [0239]aa# 5 6 7 8 S K Y F 5'-GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT GGGTTCGCCAAGC-3' (SEQ ID NO: 968) 3'-CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA CCCAAGCGGTTCG-5' (SEQ ID NO: 969)

[0240]Sets of primers may be constructed to incorporate all 18 amino acid mutations at each position in region 2. Each codon selected for mutation may be replaced with NHT, VAA or BGG in the sense direction. Exemplary primer sets for mutation of each of positions 5-8 are shown below.

[0241]Mutation of the S position (aa5) in region 2 above may be accomplished by the following primers:R2-5-NHT 5'-GCTGCTTCCGGATTCACTTT-CNHTAAGTACTTTATGTTTTGGGTTCGCCAAGC-3'(SEQ ID NO: 970); R2-5-VAA 5'-GCTGCTTCCGGATTCACTTTCVAAAAGTACTTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 971); and R2-5-BGG 5'-GCTGCTTCCGGATTCACTTTCBGGAAGTAC-TTTATGT-TTTGGGTTCGCCAAGC-3' (SEQ ID NO: 972).

[0242]Mutation of the K position (aa6) in region 2 above may be accomplished by the following primers: R2-6-NHT 5'-GCTGCTTCCGGATTCACTTTCTCTNHTTACTTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 973); R2-6-VAA 5'-GCTGCTTCCGGATTCACTTTCTCTVAATACTTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 974); and R2-6-BGG 5'-GCTGCTTCCGGATTCACTTTCTCTBGGTAC TTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 975).

[0243]Mutation of the Y position (aa7) in region 2 above may be accomplished by the following primers: R2-7-NHT 5'-GCTGCTTCCGGATTCACTTT CTCTAAGNHTTTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 976); R2-7-VAA 5'-GCTGCTTCCGGATTCACTTTCTCTAAGVAATTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 977); and R2-7-BGG 5'-GCTGCTTCCGGATTCACTTTCTCTAAGBGG TTTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 978).

[0244]Mutation of the F position (aa8) in region 2 above may be accomplished by the following primers: R2-8-NHT 5'-GCTGCTTCCGGATTCACTTT CTCTAAGTACNHTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 979); R2-8-VAA 5'-GCTGCTTCCGGATTCACTTTCTCTAAGTACVAAATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 980); and R2-8-BGG 5'-GCTGCTTCCGGATTCACTTTCTCTAAGTAC BGGATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 981).

[0245]Alternatively, modified antibody variable domains containing amino acid changes at one or more contacting (C) residues present within an exemplary antibody may be synthesized by QUIKCHANGE® site-directed mutagenesis (STRATAGENE, Texas).

[0246]In an exemplary method, QUIKCHANGE® site-directed mutagenesis may be performed to replace one or more codons in an antibody variable region (e.g., a CDR) such as XPA-23. Mutagenic primers are designed to contain the desired mutation and anneal to the same sequence on opposite strands of a plasmid comprising a nucleotide coding for XPA-23. Preferably, the desired mutation in the middle of the primer contains 20 bases of correct sequence on both sides of the nucleic acid flanking the mutation. The XPA-23 CDR1 coding region is shown below.

TABLE-US-00015 aa# 1 2 3 4 5 6 7 8 G F T F S K Y F 5'-G TCTTTCTTGC GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT (SEQ ID NO: 851) GGGTTCGCCAAGC-3' 3'-C AGAAAGAACG CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA (SEQ ID NO: 852) CCCAAGCGGTTCG-5'

[0247]Primers for QUIKCHANGE® site-directed mutagenesis are synthesized such that they are complementary to a parent nucleic acid sequence with the exception that they comprise a NHT, a VAA, or a BGG codon in the sense direction, and a ADN, a TTB, or a CCV codon in the antisense direction at the position to be mutagenized in the parent nucleic acid. Exemplary primers for mutagenesis of each of the eight amino acid residues in the XPA-23 heavy chain CDR1 are shown below and comprise a degenerate codon (underlined nucleotide triplet):

[0248]Mutation of the G position (aa1) may be accomplished by the following primers: 5'-GTCTTTCTTGCGCTGCTTCCNHTTTCACTTTCTCTAAGTACTTTATG-3' (SEQ ID NO: 853) and 3'-CAGAAAGAACGCGACGAAGGNDAAAGTGAAAGAGATTCATGAAATAC-5' (SEQ ID NO: 854); 5'-GTCTTTCTTGCGCTGCTTCCVAATTCACTTTCTCTAAGTACTTTATG-3' (SEQ ID NO: 855) and 3'-CAGAAAGAACGCGACGAAGGBTTAAGTGAAAGAGATTCATGAAATAC-5' (SEQ ID NO: 856); and 5'-GTC-TTTCTTGCGCTGCTTCCBGGTTCACTTTCTCTAAGTACTTTATG-3' (SEQ ID NO: 857) and 3'-CAGAAAGAACGCGACGAAGGVCCAAGTGAAAGAGATTCATGAAATAC-5' (SEQ ID NO: 858).

[0249]Mutation of the F position (aa2) may be accomplished by the following primers: 5'-CTTTCTTGCGCTGCTTCCGGANHTACTTTCTCTAAGTACTTTATG-3' (SEQ ID NO: 859) and 3'-GAAAGAACGCGACGAAGGCCTNDATGAAAGAGATTCATGAAATAC-5' (SEQ ID NO: 860); 5'-CTTTCTTGCGCTGCTTCCGGAVAAACTTTCTCTAAGTACTTTATG-3' (SEQ ID NO: 861) and 3'-GAAAGAACGCGACGAAGGCCTBTTTGAAAGAGATTCATGAAATAC-5' (SEQ ID NO: 862); and 5'-CTTTCTTGCGCTGCTTCCGGABGGACTTTCTCTAAGTACTTTATG-3' (SEQ ID NO: 863) and 3'-GAAAGAACGCGACGAAGGCCTVCCTGAAAGAGATTCATGAAATAC-5' (SEQ ID NO: 864).

[0250]Mutation of the T (aa3) position may be accomplished by the following primers: 5'-CTTGCGCTGCTTCCGGATTCNHTTTCTCTAAGTACTTTATGTTTTG-3' (SEQ ID NO: 865) and 3'-GAACGCGACGAAGGCCTAAGNDAAAGAGATTCATGAAATACAAAAC-5' (SEQ ID NO: 866); 5'-CTTGCGCTGCTTCCGGATTCVAATTCTCTAAGTACTTTATGTTTTG-3' (SEQ ID NO: 867) and 3'-GAACGCGACGAAGGCCTAAGBTTAAGAGATTCATGAAATACAAAAC-5' (SEQ ID NO: 868); and 5'-CTTGCGCTGCTTCCGGATTCBGGTTCTCTAAGTACTTTATGTTTTG-3' (SEQ ID NO: 869) and 3'-GAACGCGACGAAGGCCTAAGVCCAAGAGATTCATGAAATACAAAAC-5' (SEQ ID NO: 870).

[0251]Mutation of the F (aa4) position may be accomplished by the following primers: 5'-CGCTGCTTCCGGATTCACTNHTTCTAAGTACTTTATGTTTTGGG-3' (SEQ ID NO: 871) and 3'-GCGACGAAGGCCTAAGTGANDAAGATTCATGAAATACAAAACCC-5' (SEQ ID NO: 872); 5'-CGCTGCTTCCGGATTCACTVAATCTAAGTACTTTATGTTTTGGG-3' (SEQ ID NO: 873) and 3'-GCGACGAAGGCCTAAGTGABTTAGATTCATGAAATACAAAACCC-5' (SEQ ID NO: 874); and 5'-CGCTGCTTCCGGATTCACTBGGTCTAAGTACTTTATGTTTTGGG-3' (SEQ ID NO: 875) and 3'-GCGACGAAGGCCTAAGTGAVCCAGATTCATGAAATACAAAACCC-5' (SEQ ID NO: 876).

[0252]Mutation of the S (aa5) position may be accomplished by the following primers: 5'-CTGCTTCCGGATTCACTTTCNHTAAGTACTTTATGTTTTGGGTTCG-3' (SEQ ID NO: 877) and 3'-GACGAAGGCCTAAGTGAAAGNDATTCATGAAATACAAAACCCAAGC-5' (SEQ ID NO: 878); 5'-CTGCTTCCGGATTCACTTTCVAAAAGTACTTTATGTTTTGGGTTCG-3'(SEQ ID NO: 879) and 3'-GACGAAGGCCTAAGTGAAAGBTTTTCATGAAATACAAAACCCAAGC-5'(SEQ ID NO: 880); and 5'-CTGCTTCCGGATTCACTTTCBGGAAGTACTTTATGTTTTGGGTTCG-3'(SEQ ID NO: 881) and 3'-GACGAAGGCCTAAGTGAAAGVCCTTCATGAAATACAAAACCCAAGC-5'(SEQ ID NO: 882).

[0253]Mutation of the K (aa6) position may be accomplished by the following primers: 5'-CTTCCGGATTCACTTTCTCTNHTTACTTTATGTTTTGGGTTCGCC-3'(SEQ ID NO: 883) and 3'-GAAGGCCTAAGTGAAAGAGANDAATGAAATACAAAACCCAAGCGG-5'(SEQ ID NO: 884); 5'-CTTCCGGATTCACTTTCTCTVAATACTTTATGTTTTGGGTTCGCC-3'(SEQ ID NO: 885) and 3'-GAAGGCCTAAGTGAAAGAGABTTATGAAATACAAAACCCAAGCGG-5'(SEQ ID NO: 886); and 5'-CTTCCGGATTCACTTTCTCTBGGTACTTTATGTTTTGGGTTCGCC-3'(SEQ ID NO: 887) and 3'-GAAGGCCTAAGTGAAAGAGAVCCATGAAATACAAAACCCAAGCGG-5'(SEQ ID NO: 888).

[0254]Mutation of the Y (aa7) position may be accomplished by the following primers: 5'-CCGGATTCACTTTCTCTAAGNHTTTTATGTTTTGGGTTCGCCAAG-3'(SEQ ID NO: 889) and 3'-GGCCTAAGTGAAAGAGATTCNDAAAATACAAAACCCAAGCGGTTC-5'(SEQ ID NO: 890); 5'-CCGGATTCACTTTCTCTAAGVAATTTATGTTTTGGGTTCGCCMG-3'(SEQ ID NO: 891) and 3'-GGCCTAAGTGAAAGAGATTCBTTAAATACAAAACCCAAGCGGTTC-5'(SEQ ID NO: 892); and 5'-CCGGATTCACTTTCTCTAAGBGGTTTATGTTTTGGGTTCGCCAAG-3'(SEQ ID NO: 893) and 3'-GGCCTAAGTGAAAGAGATTCVCCAAATACAAAACCCAAGCGGTTC-5'(SEQ ID NO: 894).

[0255]Mutation of the F (aa8) position may be accomplished by the following primers: 5'-GGATTCACTTTCTCTAAGTACNHTATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 895) and 3'-CCTAAGTGAAAGAGATTCATGNDATACAAAACCCAAGCGGTTCG-5' (SEQ ID NO: 896); 5'-GGATTCACTTTCTCTAAGTACVAAATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 897) and 3'-CCTAAGTGAAAGAGATTCATGBTTTACAAAACCCAAGCGGTTCG-5' (SEQ ID NO: 898); and 5'-GGATTCACTTTCTCTAAGTACBGGATGTTTTGGGTTCGCCAAGC-3' (SEQ ID NO: 899) and 3'-CCTAAGTGAAAGAGATTCATGVCCTACAAAACCCAAGCGGTTCG-5' (SEQ ID NO: 900).

C. Synthesis of Full-Length Mutagenized Antibody

[0256]Full-length mutagenized antibodies may be produced by recombinant DNA technologies.

[0257]For the PCR-based method, a first PCR reaction (PCR1) is performed with a R2-rev primer and a 5'-Sfil primer, which incorporates a 5' Sfil restriction site into the amplified fragment. For each library oligonucleotide containing the mutations described above, the PCR2 reaction is performed to create the DNA fragment incorporating the primer mutation and the 3' Sfil restriction site. For the mutations in region 2, twelve PCR2 reactions will be performed with forward primers denoted R2-5 through R2-8 above (denoted primer-F in PCR2 below). The reverse primer for the mutagenic reaction will be 3'-Sfil. An appropriate amount of the following reagents may be used for PCR1: PfuUltra buffer; dNTPs [10 μM], template (10 ng total), 5'-Sfil [25 pmol], R2-rev [25 pmol], PfuUltra (2.5 U/μL), dH2O to 50 μL total. An appropriate amount of the following reagents may be used for PCR2: PfuUltra buffer, dNTPs [10 μM], template (10 ng total), Primer-F [10 pmol], 3'-Sfil [25 pmol], PfuUltra (2.5 U/μL), dH2O to 50 μL total. PCR1 and PCR2 may be conduced according to standard protocols including an initial denatural step, a number of cycles including a denaturation, annealing and extension step and a final extension step for appropriate times and temperatures.

[0258]A full-length antibody fragment may be produced by performing a separate reaction for each PCR2 product. For this step, an approximately equimolar amount of PCR product 1 and 2 is combined (e.g., 0.5 microliters of each PCR is combined). An appropriate amount of the following reagents may be used generation of a full-length antibody fragment: PfuUltra buffer, dNTPs [10 μM], PCR1 product, PCR2 product, PfuUltra (2.5 U/μL), dH2O to 50 μL total. PCR may be conduced according to standard protocols including an initial denatural step, a number of cycles including a denaturation, annealing and extension step for appropriate times and temperatures.

[0259]The full-length fragment may then be amplified by directly adding to the above reaction an appropriate amount of the following reagents: PfuUltra buffer, dNTPs [10 μM], 5'-Sfil [25 pmol], 3'-Sfil [25 pmol], PfuUltra (2.5 U/μL), dH2O to 50 μL total. PCR may be conduced according to standard protocols including an initial denaturation step, a number of cycles that comprise a denaturation, annealing and extension step for appropriate times and temperatures and a final extension step. The PCR product may be examined on an agarose gel to ensure that the amplified DNA segment is the correct length.

[0260]Next, a vector and the DNA inserts obtained from the above PCR are digested with Sfil (NEB) according to the manufacturer's instructions and gel purified. The DNA synthesized fragment may be cloned into a pXOMA Fab or pXOMA Fab-gIII vector. Briefly, the DNA fragment is purified by using the QIAGEN® PCR purification kit and sequentially digesting the fragment with Notl (NEW ENGLAND BIOLABS® Ipswich, Mass.) and Ascl (NEW ENGLAND BIOLABS® Ipswich, Mass.) (See, Methods in Molecular Biology, vol. 178: Antibody Phage Display: Methods and Protocols Edited by: P. M. O'Brien and R. Aitken, Humana Press, "Standard Protocols for the Construction of Fab Libraries, Clark, M. A., 39-58) (see, e.g., FIG. 6). Next, the vectors may be ligated with the mutagenized insert using T4 Ligase (NEW ENGLAND BIOLABS® Ipswich, Mass.) and transformed into TG1 cells by electroporation.

[0261]Alternatively, for the DPN-based method, a double-stranded DNA (e.g., dsDNA) vector with an antibody insert isolated from a dam+ host is used as template for mutagenesis. DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpnl digestion. Two synthetic oligonucleotide primers containing the desired mutation each complementary to opposite strands of the vector, are extended during temperature cycling by DNA polymerase (e.g., PfuTurbo). PCR reactions may comprise an appropriate amount of PfuUltra buffer, dNTPs [10 mM] each dNTP, template (50 ng total), Primer-F [5 μM], Primer-R [5 μM], PfuUltra (2.5 U/μL), DMSO, and dH2O up to 50 μL total and be conducted with the following cycling parameters: an initial denaturation, subsequent cycles of denaturation, annealing and extension and a final extension step. Incorporation of the mutagenesis primers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the PCR product is treated with Dpnl and incubated at an appropriate temperature (e.g., at 37° C. for 4-5 hours). The Dpnl endonuclease (target sequence: 5'-Gm6ATC-3') is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation-containing synthesized DNA. The nicked vector DNA containing the desired mutations is then transformed into supercompetent cells (e.g., XL1-Blue).

D. Sequencing of Mutagenized Antibodies

[0262]A library of mutagenized antibodies may comprise each of 18 unique amino acid mutations at each position mutated. To identify all possible unique mutations an appropriate number of clones obtained from each degenerate codon are analyzed. For example, the NHT codon encodes 12 amino acids such that 72 clones from this reaction are sequenced for each mutated position. The VAA codon encodes 3 amino acids such that 12 clones are sequenced from this reaction for each mutated position. The BGG codon encodes 3 amino acids such that 12 clones from this reaction are sequenced for each mutated position. Unique clones are rearrayed into 96-well plates.

E. Expression of Mutagenized Antibodies

[0263]Mutagenized antibodies may be expressed. In an exemplary method, starting cultures may be produced by filling a plate (e.g., a 96 well plate) with an appropriate growth media (e.g., 2YTAG (2YT+2% glucose+100 μgs/ml Ampicillin) and inoculating the plate with glycerol stocks of the mutagenized antibodies. The cultures are then grown overnight (e.g., in an ATR plate shaker incubator at 37° C. with shaking at 450 rpm). Next, plates are filled with an appropriate growth medium (e.g., 1.2 mL per well of Superbroth+100 μgs.ml Ampicillin+0.2% glucose). The plates are then Inoculated with an appropriate amount of the overnight culture (e.g., 25 μL of overnight culture). The cultures are then grown with incubation (e.g., ATR plate shaker incubator at 37° C.) and shaking (e.g., at 700 rpm until Abs600 nm=1.5). Expression in the cultures is then induced (e.g., by adding 12 uL of 100 mM IPTG per well to get a final concentration of 1 mM IPTG final) and incubated overnight (e.g., in an ATR plate shaker incubator at 30° C. with shaking at 700 rpm). Next, the plates are spun (e.g., at 4000 rpm using Beckman Coulter table top centrifuge for 10 minutes) and the supernatant decanted. The cells are then vortexed to disturb and loosen the pellet. The pellets are resuspended (e.g., with 75 μL per well of cold PPB) and incubated one ice (e.g., for 10 minutes). Next, water (e.g., 225 μL per well) is added and the cells resuspended. The suspension is incubated on ice (e.g., for 1 hour) and the plates are then spun (e.g., at 4000 rpm using Beckman Coulter table top centrifuge for 20 minutes). Last, the supernatants are collected for use in assays as described in detail below.

F. ELISA Screening of Mutagenized Antibodies

[0264]An assay including, for example, an ELISA may be performed to ensure that the mutagenized antibodies are capable of binding to their respective antigen.

[0265]In an exemplary ELISA, plates (e.g., 96-well Nunc Maxisorp plates) are coated with an antibody to the mutagenized antibody (e.g., 50 μL per well of 1 μg/ml Goat anti Human IgG (Fab)₂ Jackson immunoresearch, Cat. 109-005-006) and the plates are then incubated overnight at 4° C. After incubation, the plates may be washed (e.g., 3× with PBS-Tween at 350 μL/well) and then blocked (e.g., by adding 350 μL/well with 5% Milk+PBS).

[0266]Next, periplasmic extracts (PPE) containing the mutagenized antibody are blocked (e.g., by milk(diluted in PBS) to 200 μL of PPE to get a final milk percent of 5%). The PPEs are then mixed and incubated (e.g., at room temperature still for 1 hour) before using as samples to screen on ELISA and then washed (e.g., 3× with PBS-Tween at 350 μL/well). The blocked PPE samples (e.g., 50 μL) are then added to the blocked ELISA plates and incubated (e.g., at room temperature for 1-2 hours). Again the PPEs are washed (e.g., 3× with PBS-Tween at 350 μL/well). Next, an antibody specific for the mutagenized antibody is added to the PPEs (e.g., 50 μL/well of 1 μg/ml monoclonal anti-V5 antibody, Sigma Cat.#V8012-50UG) and the PPEs incubated (e.g., at room temperature for 1 hour). Again the PPEs are washed (e.g., 3× with PBS-Tween at 350 μL/well). Next, a secondary antibody conjugated to a enzymatic label is added to the PPEs (e.g., 1:10000 diluted Goat anti mouse HRP conjugated, Biorad, Cat. 170-5047) and incubated with the PPEs (e.g., for 1 hour at room temperature). Again the PPEs are washed (e.g., 3× with PBS-Tween at 350 μL/well). Next, an appropriate amount of substrate for the enzymatic label is added to the PPEs (e.g., 50 μL/well of TMB, soluble, Calbiochem, Cat. 613544) and the enzyme is allowed time to act on the substrate (e.g., until sufficiently blue color develops). The reaction may be stopped by the addition of an agent that sequesters the substrate and/or and agent that inhibits the enzymatic activity of the secondary antibody (e.g., 50 μL per well of 2N H₂SO₄). Last, absorbance of the samples are read at 450 nm.

G. Ranking of Mutagenized Antibodies

[0267]Mutagenized antibodies may be ranked based on their dissociation rate from their respective antigen.

[0268]In an exemplary method, a Biacore A100 screening protocol may be used to rank mutagenized antibody clones. For example, a CM5 chip may be docked and normalized using normalization solution (e.g., using A100 normalization solution and use and an appropriate running buffer (e.g., HBS-N (0.01 M HEPES pH 7.4, 0.15 M NaCl). After normalization, software is set to immobilize antigen on desired spots of each flow cell. For antigen surface preparation the surface may be activated (e.g., with NHS/EDC mixture from the amine coupling kit for 5 minutes at 10 μl/min). Antigen is then diluted (e.g., in 10 mM sodium acetate buffer) and the surface of the CM5 chip is blocked (e.g., with 1 M ethanolamine HCl pH 8.5 for 5 min at 10 μl/min). Next, each sample comprising a mutagenized antibody is injected over the CM5 chip (e.g., for 3 min at 30 μl/min flow rate with 600 s dissociation) at an appropriate temperature (e.g., 25° C.). Biaevaluation software (e.g., Biacore A100 evaluation software) is then used to calculate dissociation rates of individual samples and the relative amount of sample bound to each test surface. The data is fit to an appropriate kinetic model (e.g., the kinetic titration model).

Embodiments

[0269]1. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain a modified variable domain with enhanced binding affinity to the binding partner, the method comprising: [0270]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0271]b. substituting one or more contacting (C) amino acid residues with other amino acid residues, thereby generating a library of modified variable domains; [0272]c. screening the library for binding affinity to the binding partner; and [0273]d. obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0274]2. The method of embodiment 1, wherein each contacting (C) residue is substituted.

[0275]3. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain a modified variable domain with enhanced binding affinity to the binding partner, the method comprising: [0276]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0277]b. substituting one or more peripheral (P) amino acid residues with other amino acid residues, thereby generating a library of modified variable domains; [0278]c. screening the library for binding affinity to the binding partner; and [0279]d. obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0280]4. The method of embodiment 3, wherein each peripheral (P) residue is substituted.

[0281]5. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain a modified variable domain with enhanced binding affinity to the binding partner, the method comprising: [0282]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0283]b. substituting one or more supporting (S) amino acid residues with other amino acid residues, thereby generating a library of modified variable domains; [0284]c. screening the-library-for-binding-affinity-to-the-binding-partner; and [0285]d. obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0286]6. The method of embodiment 5, wherein each supporting (S) residue is substituted.

[0287]7. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain a modified variable domain with enhanced binding affinity to the binding partner, the method comprising: [0288]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0289]b. substituting one or more interfacial (I) amino acid residues with other amino acid residues, thereby generating a library of modified variable domains; [0290]c. screening the library for binding affinity to the binding partner; and [0291]d. obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0292]8. The method of embodiment 7, wherein each interfacial (I) residue is substituted.

[0293]9. The method of embodiment 1, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0294]10. The method of embodiment 9, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0295]11. The method of embodiment 1, wherein the contacting residue is in CDR2 in a light chain variable domain.

[0296]12. The method of embodiment 11, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0297]13. The method of embodiment 1, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0298]14. The method of embodiment 13, wherein the contacting residue is at position 32 or 33 in CDR1.

[0299]15. The method of embodiment 1, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0300]16. The method of embodiment 15, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0301]17. The method of any one of embodiments 1, 3, 5 or 7, wherein the other amino acid residues are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0302]18. The method of any one of embodiments 1, 3, 5 or 7, wherein the other amino acid substitutions are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0303]19. The method of any one of embodiments 1, 3, 5 or 7, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0304]20. The method of any one of claim 1, 3, 5 or 7, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0305]21. The method of any one of embodiments 1, 3, 5 or 7, wherein the variable domain is from a humanized antibody.

[0306]22. The method of any one of embodiments 1, 3, 5 or 7, wherein the variable domain is from a human antibody.

[0307]23. The method of any one of embodiments 1, 3, 5 or 7, wherein binding affinity is determined by measuring K_off.

[0308]24. A method of making a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0309]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode a contacting (C) residue identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at the C residue thereby generating a library of modified antibody variable domains; and [0310]b. selecting a modified variable domain from the library that has enhanced binding affinity to the binding partner compared to the parent variable domain.

[0311]25. The method of embodiment 24, wherein each contacting (C) residue is substituted.

[0312]26. A method of making a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0313]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode a peripheral (P) residue identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at the P residue, thereby generating a library of modified antibody variable domains; and [0314]b. selecting a modified variable domain from the library that has enhanced binding affinity to the binding partner compared to the parent variable domain.

[0315]27. The method of embodiment 1, wherein each peripheral (P) residue is substituted.

[0316]28. A method of making a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0317]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode a supporting (S) residue identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at the S residue thereby generating a library of modified antibody variable domains; and [0318]b. selecting a modified variable domain from the library that has enhanced binding affinity to the binding partner compared to the parent variable domain.

[0319]29. The method of embodiment 28, wherein each supporting (S) residue is substituted.

[0320]30. A method of making a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0321]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode an interfacial (I) residue identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at the I residue, thereby generating a library of modified antibody variable domains; and [0322]b. selecting the modified variable domain from the library that has enhanced binding affinity to a binding partner compared to a parent variable domain compared to the parent variable domain.

[0323]31. The method of embodiment 30, wherein each interfacial (I) residue is substituted.

[0324]32. The method of embodiment 24, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0325]33. The method of embodiment 32, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0326]34. The method of embodiment 24 wherein the contacting residue is in CDR2 in a light chain variable domain.

[0327]35. The method of embodiment 34, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0328]36. The method of embodiment 24, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0329]37. The method of embodiment 36, wherein the contacting residue is at position 32 or 33 in CDR1.

[0330]38. The method of embodiment 24, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0331]39. The method of embodiment 38, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0332]40. The method of any one of embodiments 24, 26, 28 or 30 further comprising inserting the modified antibody variable domain into an appropriate vector.

[0333]41. The method of embodiment 40, wherein the vector is either a plasmid or a phage.

[0334]42. The method of any one of embodiment 41, wherein the vector is pXOMA Fab or pXOMA Fab-gIII.

[0335]43. The method of any one of embodiments 24, 26, 28 or 30, wherein the amino acid substitutions are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0336]44. The method of any one of embodiments 24, 26, 28 or 30, wherein the amino acid substitutions are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0337]45. The method of any one of embodiments 24, 26, 28 or 30, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0338]46. The method of any one of embodiments 24, 26, 28 or 30, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0339]47. The method of any one of embodiments 24, 26, 28 or 30, wherein the variable domain is from a humanized antibody.

[0340]48. The method of any one of embodiments 24, 26, 28 or 30, wherein the variable domain is from a human antibody.

[0341]49. The method of any one of embodiments 24, 26, 28 or 30, wherein binding affinity is determined by measuring K_off.

[0342]50. The method of any one of embodiments 24, 26, 28 or 30, wherein step (b) comprises: [0343]a. contacting a parent variable domain with the binding partner under conditions that permit binding; [0344]b. contacting the modified variable domains with binding partner under conditions that permit binding; and [0345]c. determining binding affinity of the modified variable domains and the parent variable domain for the binding partner,

[0346]wherein modified variable domains that have a binding affinity for the binding partner greater than the binding affinity of the parent variable domain for the binding partner are identified as having enhanced binding affinity for the binding partner.

[0347]51. A method for selecting a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0348]a. obtaining a library of modified antibody variable domains comprising amino acid substitutions at a contacting (C) residue identified from the "prox" line as shown in FIGS. 3A or 3B; [0349]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0350]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0351]52. A method for selecting a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0352]a. obtaining a library of modified antibody variable domains comprising amino acid substitutions at a peripheral (P) residue identified from the "prox" line as shown in FIGS. 3A or 3B; [0353]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0354]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0355]53. A method for selecting a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0356]a. obtaining a library of modified antibody variable domains comprising amino acid substitutions at a supporting (S) residue identified from the "prox" line as shown in FIGS. 3A or 3B; [0357]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0358]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0359]54. A method for selecting a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0360]a. obtaining a library of modified antibody variable domains comprising amino acid substitutions at an interfacial (I) residue identified from the "prox" line as shown in FIGS. 3A or 3B; [0361]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0362]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0363]55. The method of embodiment 51, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0364]56. The method of embodiment 55, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0365]57. The method of embodiment 51, wherein the contacting residue is in CDR2 in a light chain variable domain.

[0366]58. The method of embodiment 57, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0367]59. The method of embodiment 51, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0368]60. The method of embodiment 59, wherein the contacting residue is at position 32 or 33 in CDR1.

[0369]61. The method of embodiment 51, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0370]62. The method of embodiment 61, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0371]63. The method of any one of embodiments 51 to 54, wherein the amino acid substitutions are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0372]64. The method of any one of embodiments 51 to 54, wherein the amino acid substitutions are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0373]65. The method of any one of embodiments 51 to 54, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0374]66. The method of any one of embodiments 51 to 54, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/GfT, D=A/G/T).

[0375]67. The method of any one of embodiments 51 to 54, wherein the variable domain is from a humanized antibody.

[0376]68. The method of any one of embodiments 51 to 54, wherein the variable domain is from a human antibody.

[0377]69. The method of any one of embodiments 51 to 54, wherein binding affinity is determined by measuring K_off.

[0378]70. A method of producing a nucleic acid library with an equal representation of one or more non-redundant amino acid changes at each of one or more positions in a parent nucleic acid, the method comprising: [0379]a. providing a set of primers that each comprise at least one degenerate codon at identical positions, wherein the primers are complementary to a sequence in the parent nucleic acid and the primers code for an equal representation of non-redundant amino acid changes at one or more positions; [0380]b. hybridizing a primer from the set to the parent nucleic acid; [0381]c. amplifying the parent nucleic acid molecule with the primer to generate one or more nucleic acids that code for amino acid changes at one or more identical positions; [0382]d. repeating steps (b) and (c) with remaining primers from the set; [0383]e. pooling the nucleic acids produced with each primer in step (d); and [0384]f. obtaining a library of nucleic acids coding for an equal representation of one or more amino acid changes at one or more identical positions, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0385]71. The method of embodiment 70, wherein the primer set codes for eighteen amino acid changes at each of one or more positions in the parent nucleic acid.

[0386]72. The method of embodiment 71, wherein the set of primers comprises three primers.

[0387]73. The method of embodiment 71, wherein the set of primers comprises seven primers.

[0388]74. The method of embodiment 72 or 73, wherein the primers each comprise a degenerate codon which collectively code for alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine at each position.

[0389]75. The method of embodiment 72, wherein the primers each comprise one or more degenerate codons as represented by NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0390]76. The method of embodiment 73, wherein the primers each comprise one or more degenerate codons as represented by ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

[0391]77. The method of embodiment 70, wherein the primer set codes for basic amino acid changes at each of one or more positions in the parent nucleic acid.

[0392]78. The method of embodiment 77, wherein the primer set comprises one primer.

[0393]79. The method of embodiment 78, wherein the one primer comprises a degenerate codon which codes for arginine and lysine.

[0394]80. The method of embodiment 79, wherein the one primer comprises one or more degenerate codons as represented by ARG (where, R=A/G).

[0395]81. The method of embodiment 70, wherein the primer set codes for polar amino acid changes at each of one or more positions in the parent nucleic acid.

[0396]82. The method of embodiment 81, wherein the primer set comprises two primers.

[0397]83. The method of embodiment 82, wherein the two primers each comprise a degenerate codon which collectively code for serine, threonine, asparagine and tyrosine.

[0398]84. The method of embodiment 83, wherein the two primers each comprise one or more degenerate codons as represented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

[0399]85. The method of embodiment 70, wherein the primer set codes for acidic amino acid changes at each of one or more positions in the parent nucleic acid.

[0400]86. The method of embodiment 85, wherein the primer set comprises one degenerate codon.

[0401]87. The method of embodiment 86, wherein the one primer comprises a degenerate codon that codes for glutamic acid and aspartic acid.

[0402]88. The method of embodiment 87, wherein the one primer comprises one or more degenerate codons as represented by GAS (where S=C/G).

[0403]89. The method of embodiment 70, wherein the primers code for non-polar amino acid changes at each of one or more positions in the parent nucleic acid.

[0404]90. The method of embodiment 89, wherein the primer set comprises three degenerate codons.

[0405]91. The method of embodiment 90, wherein the three primers each comprise a degenerate codon that collectively code for glutamic acid and aspartic acid.

[0406]92. The method of embodiment 91, wherein the primers each comprise one or more degenerate codons as represented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G).

[0407]93. The method of embodiment 70, where the parent nucleic acid encodes an antibody variable region.

[0408]94. The method of embodiment 70, wherein the positions in the parent nucleic acid code for contacting (C) residues.

[0409]95. A set of primers comprising: [0410]at least one degenerate codon at identical positions, wherein the degenerate codons code for an equal representation of one or more non-redundant amino acid changes at each of one or more positions in the parent nucleic acid and the primers are complementary to a sequence in the parent nucleic acid, with the proviso that the degenerate codons do not code for methionine or cysteine.

[0411]96. The set of primers of embodiment 95, wherein the primer set codes for eighteen amino acid changes at each of one or more positions in the parent nucleic acid.

[0412]97. The set of primers of embodiment 96, wherein the set of primers comprises three primers.

[0413]98. The set of primers of embodiment 96, wherein the set of primers comprises seven primers.

[0414]99. The set of primers of embodiment 97 or 98, wherein the primers each comprise a degenerate codon which collectively code for alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine at each position.

[0415]100. The method of embodiment 97, wherein the primers each comprise one or more degenerate codons as represented by NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0416]101. The set of primers of embodiment 98, wherein the primers each comprise one or more degenerate codons as represented by ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

[0417]102. The set of primers embodiment 95, wherein the primer set codes for basic amino acid changes at each of one or more positions in the parent nucleic acid.

[0418]103. The set of primers of embodiment 102, wherein the primer set comprises one primer.

[0419]104. The set of primers of embodiment 103, wherein the one primer comprises a degenerate codon which codes for arginine and lysine.

[0420]105. The set of primers of embodiment 104, wherein the one primer comprises one or more degenerate codons as represented by ARG (where, R=A/G).

[0421]106. The set of primers of embodiment 95, wherein the primer set codes for polar amino acid changes at each of one or more positions in the parent nucleic acid.

[0422]107. The set of primers of embodiment 106, wherein the primer set comprises two primers.

[0423]108. The set of primers of embodiment 107, wherein the two primers each comprise a degenerate codon which collectively code for serine, threonine, asparagine and tyrosine.

[0424]109. The set of primers of embodiment 108, wherein the two primers each comprise one or more degenerate codons as represented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

[0425]110. The set of primers of embodiment 95, wherein the primer set codes for acidic amino acid changes at each of one or more positions in the parent nucleic acid.

[0426]111. The set of primers of embodiment 110, wherein the primer set comprises one degenerate codon.

[0427]112. The set of primers of embodiment 111, wherein the one primer comprises a degenerate codon that codes for glutamic acid and aspartic acid.

[0428]113. The set of primers of embodiment 112, wherein the one primer comprises one or more degenerate codons as represented by GAS (where S=C/G).

[0429]114. The set of primers of embodiment 95, wherein the primers code for non-polar amino acid changes at each of one or more positions in the parent nucleic acid.

[0430]115. The set of primers of embodiment 114, wherein the primer set comprises three degenerate codons.

[0431]116. The set of primers of embodiment 115, wherein the three primers each comprise a degenerate codon that collectively code for glutamic acid and aspartic acid.

[0432]117. The set of primers of embodiment 116, wherein the primers each comprise one or more degenerate codons as represented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G).

[0433]118. The set of primers of embodiment 95, where the parent nucleic acid encodes an antibody variable region.

[0434]119. The set of primers of embodiment 95, wherein the positions in the parent nucleic acid code for contacting (C) residues.

[0435]120. A kit for mutagenesis of one or more positions in a parent nucleic acid, the kit comprising: [0436]a set of primers comprising at least one degenerate codon at identical positions, wherein the degenerate codons code for an equal representation of one or more non-redundant amino acid changes at each of one or more positions in the parent nucleic acid and the primers are complementary to a sequence in the parent nucleic acid, [0437]with the proviso that the degenerate codons do not code for methionine or cysteine.

[0438]121. The kit of embodiment 121, wherein the primer set codes for eighteen amino acid changes at each of one or more positions in the parent nucleic acid.

[0439]122. The kit of embodiment 121, wherein the set of primers comprises three primers.

[0440]123. The kit of embodiment 121, wherein the set of primers comprises seven primers.

[0441]124. The kit of embodiment 122 or 123, wherein the primers each comprise a degenerate codon which collectively code for alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine at each position.

[0442]125. The kit of embodiment 122, wherein the primers each comprise one or more degenerate codons as represented by NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0443]126. The kit of embodiment 123, wherein the primers each comprise one or more degenerate codons as represented by ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

[0444]127. The kit of embodiment 120, wherein the primer set codes for basic amino acid changes at each of one or more positions in the parent nucleic acid.

[0445]128. The kit of embodiment 127, wherein the primer set comprises one primer.

[0446]129. The kit of embodiment 128, wherein the one primer comprises a degenerate codon which codes for arginine and lysine.

[0447]130. The kit of embodiment 129, wherein the one primer comprises one or more degenerate codons as represented by ARG (where, R=A/G). 131. The kit of embodiment 120, wherein the primer set codes for polar amino acid changes at each of one or more positions in the parent nucleic acid.

[0448]132. The kit of embodiment 131, wherein the primer set comprises two primers.

[0449]133. The kit of embodiment 132, wherein the two primers each comprise a degenerate codon which collectively code for serine, threonine, asparagine and tyrosine.

[0450]134. The kit of embodiment 133, wherein the two primers each comprise one or more degenerate codons as represented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

[0451]135. The kit of embodiment 120, wherein the primer set codes for acidic amino acid changes at each of one or more positions in the parent nucleic acid.

[0452]136. The kit of embodiment 135, wherein the primer set comprises one degenerate codon.

[0453]137. The kit of embodiment 136, wherein the one primer comprises a degenerate codon that codes for glutamic acid and aspartic acid.

[0454]138. The kit of embodiment 137, wherein the one primer comprises one or more degenerate codons as represented by GAS (where S=C/G).

[0455]139. The kit of embodiment 120, wherein the primers code for non-polar amino acid changes at each of one or more positions in the parent nucleic acid.

[0456]140. The kit of embodiment 139, wherein the primer set comprises three degenerate codons.

[0457]141. The kit of embodiment 140, wherein the three primers each comprise a degenerate codon that collectively code for glutamic acid and aspartic acid.

[0458]142. The kit of embodiment 141, wherein the primers each comprise one or more degenerate codons as represented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G).

[0459]143. The kit of embodiment 120, where the parent nucleic acid encodes an antibody variable region.

[0460]144. The kit of embodiment 120, wherein the positions in the parent nucleic acid code for contacting (C) residues.

[0461]145. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain a modified variable domain with enhanced binding affinity to the binding partner, the method comprising: [0462]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0463]b. substituting one or more contacting (C), peripheral (P), supporting (S) and interfacial (I) amino acid residues with other amino acid residues, thereby generating a library of modified variable domains; [0464]c. screening the library for binding affinity to the binding partner; and [0465]d. obtaining a modified variable domain with enhanced binding affinity to the binding partner.

[0466]146. The method of embodiment 145, wherein each contacting (C), peripheral (P), supporting (S) and interfacial (I) residue is substituted.

[0467]147. A method of making a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0468]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode a contacting (C), peripheral (P), supporting (S) and interfacial (I) residue identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at the C residue thereby generating a library of modified antibody variable domains; and [0469]b. selecting a modified variable domain from the library that has enhanced binding affinity to the binding partner compared to the parent variable domain.

[0470]148. The method of embodiment 24, wherein each contacting (C), peripheral (P), supporting (S) and interfacial (I) residue is substituted.

[0471]149. A method for selecting a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0472]a. obtaining a library of modified antibody variable domains comprising amino acid substitutions at one or more contacting (C), peripheral (P), supporting (S) and interfacial (I) residues identified from the "prox" line as shown in FIGS. 3A or 3B; [0473]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0474]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0475]150. The method of embodiment 149, wherein each contacting (C), peripheral (P), supporting (S) and interfacial (I) residue is substituted.

[0476]1A. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain modified variable domains with enhanced binding affinity to the binding partner, the method comprising: [0477]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0478]b. substituting one or more contacting (C) amino acid residues in the antibody variable domain with other amino acid residues to generate an array of modified variable domains; [0479]c. screening the array of modified variable domains for binding affinity to the binding partner; and [0480]d. obtaining modified variable domains with enhanced binding affinity to the binding partner.

[0481]2A. The method of embodiment 1, wherein each contacting (C) residue in the antibody variable domain is separately substituted.

[0482]3A. The method of embodiment 1, wherein one or more contacting (C) residues in the antibody variable domain are simultaneously substituted.

[0483]4A. The method of embodiment 1, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0484]5A. The method of embodiment 4, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0485]6A. The method of embodiment 1, wherein the contacting residue is in CDR2 in a light chain variable domain.

[0486]7A. The method of embodiment 6, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0487]8A. The method of embodiment 1, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0488]9A. The method of embodiment 8, wherein the contacting residue is at position 32 or 33 in CDR1.

[0489]10A. The method of embodiment 1, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0490]11A. The method of embodiment 10, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0491]12A. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain modified variable domains with enhanced binding affinity to the binding partner, the method comprising: [0492]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0493]b. substituting one or more peripheral (P) amino acid residues in the antibody variable domain with other amino acid residues to generate an array of modified variable domains; [0494]c. screening the array of modified variable domains for binding affinity to the binding partner; and [0495]d. obtaining modified variable domains with enhanced binding affinity to the binding partner.

[0496]13A. The method of embodiment 12, wherein each peripheral (P) residue in the antibody variable domain is separately substituted.

[0497]14A. The method of embodiment 12, wherein one or more peripheral (P) residues in the antibody variable domain are simultaneously substituted.

[0498]15A. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain modified variable domains with enhanced binding affinity to the binding partner, the method comprising: [0499]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0500]b. substituting one or more supporting (S) amino acid residues in the antibody variable domain with other amino acid residues to generate an array of modified variable domains; [0501]c. screening the array of modified variable domains for binding affinity to the binding partner; and [0502]d. obtaining modified variable domains with enhanced binding affinity to the binding partner.

[0503]16A. The method of embodiment 15, wherein each supporting (S) residue in the antibody variable domain is separately substituted.

[0504]17A. The method of embodiment 15, wherein one or more supporting (S) residues in the antibody variable domain are simultaneously substituted.

[0505]18A. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain modified variable domains with enhanced binding affinity to the binding partner, the method comprising: [0506]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0507]b. substituting one or more interfacial (I) amino acid residues in the antibody variable domain with other amino acid residues to generate an array of modified variable domains; [0508]c. screening the array of modified variable domains for binding affinity to the binding partner; and [0509]d. obtaining modified variable domains with enhanced binding affinity to the binding partner.

[0510]19A. The method of embodiment 1, wherein each interfacial (I) residue in the antibody variable domain is separately substituted.

[0511]20A. The method of embodiment 1, wherein one or more interfacial (I) residues in the antibody variable domain are simultaneously substituted.

[0512]21A. The method of any one of embodiments 1, 12, 15 or 18, wherein the other amino acid residues are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0513]22A. The method of any one of embodiments 1, 12, 15 or 18, wherein the other amino acid substitutions are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0514]23A. The method of embodiment 22, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0515]24A. The method of embodiment 22, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=NC/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0516]25A. The method of any one of embodiments 1, 12, 15 or 18, wherein the variable domain is from a humanized antibody.

[0517]26A. The method of any one of embodiments 1, 12, 15 or 18, wherein the variable domain is from a human antibody.

[0518]27A. The method of any one of embodiments 1, 12, 15 or 18, wherein binding affinity is determined by measuring K_off.

[0519]28A. The method of making modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0520]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode contacting (C) residues identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at C residues to generate an array of modified antibody variable domains; and [0521]b. selecting modified variable domains from the array that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0522]29A. The method of embodiment 28, wherein each contacting (C) residue in the antibody variable domain is separately substituted.

[0523]30A. The method of embodiment 28, wherein one or more contacting (C) residues in the antibody variable domain are simultaneously substituted.

[0524]31A. The method of embodiment 28, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0525]32A. The method of embodiment 31, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0526]33A. The method of embodiment 28, wherein the contacting residue is in CDR2 in a light chain variable domain.

[0527]34A. The method of embodiment 33, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0528]35A. The method of embodiment 28, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0529]36A. The method of embodiment 35, wherein the contacting residue is at position 32 or 33 in CDR1.

[0530]37A. The method of embodiment 28, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0531]38A. The method of embodiment 37, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0532]39A. A method of making modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0533]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode peripheral (P) residues identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at P residues to generate an array of modified antibody variable domains; and [0534]b. selecting modified variable domains from the array that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0535]40A. The method of embodiment 39, wherein each peripheral (P) residue in the antibody variable domain is separately substituted.

[0536]41A. The method of embodiment 39, wherein one or more peripheral (P) residues in the antibody variable domain are simultaneously substituted.

[0537]42A. A method of making modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0538]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode supporting (S) residues identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at S residues to generate an array of modified antibody variable domains; and [0539]b. selecting modified variable domains from the array that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0540]43A. The method of embodiment 42, wherein each supporting (S) residue in the antibody variable domain is separately substituted.

[0541]44A. The method of embodiment 42, wherein one or more supporting (S) residues in the antibody variable domain are simultaneously substituted.

[0542]45A. A method of making modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0543]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode interfacial (I) residues identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at I residues to generate an array of modified antibody variable domains; and [0544]b. selecting modified variable domains from the array that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0545]46A. The method of embodiment 45, wherein each interfacial (I) residue in the antibody variable domain is separately substituted.

[0546]47A. The method of embodiment 45, wherein one or more interfacial (I) residues in the antibody variable domain are simultaneously substituted.

[0547]48A. The method of any one of embodiments 28, 39, 42 or 45 further comprising inserting the modified antibody variable domains into an appropriate vector.

[0548]49A. The method of embodiment 48, wherein the vector is either a plasmid or a phage.

[0549]50A. The method of any one of embodiment 49, wherein the vector is pXOMA Fab or pXOMA Fab-gIII.

[0550]51A. The method of any one of embodiments 28, 39, 42 or 45, wherein the amino acid substitutions are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0551]52A. The method of any one of embodiments 28, 39, 42 or 45, wherein the amino acid substitutions are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0552]53A. The method of embodiment 52, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0553]54A. The method of embodiment 52, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0554]55A. The method of any one of embodiments 28, 39, 42 or 45, wherein the variable domain is from a humanized antibody.

[0555]56A. The method of any one of embodiments 28, 39, 42 or 45, wherein the variable domain is from a human antibody.

[0556]57A. The method of any one of embodiments 28, 39, 42 or 45, wherein binding affinity is determined by measuring K_off.

[0557]58A. The method of any one of embodiments 28, 39, 42 or 45, wherein step (b) comprises: [0558]a. contacting a parent variable domain with the binding partner under conditions that permit binding; [0559]b. contacting the modified variable domains with binding partner under conditions that permit binding; and [0560]c. determining binding affinity of the modified variable domains and the parent variable domain for the binding partner,

[0561]wherein modified variable domains that have a binding affinity for the binding partner greater than the binding affinity of the parent variable domain for the binding partner are identified as having enhanced binding affinity for the binding partner.

[0562]59A. A method for selecting modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0563]a. obtaining an array of modified antibody variable domains comprising amino acid substitutions at one or more contacting (C) residues identified from the "prox" line as shown in FIGS. 3A or 3B; [0564]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0565]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0566]60A. A method for selecting modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0567]a. obtaining an array of modified antibody variable domains comprising amino acid substitutions at one or more peripheral (P) residues identified from the "prox" line as shown in FIGS. 3A or 3B; [0568]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0569]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0570]61A. A method for selecting modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0571]a. obtaining an array of modified antibody variable domains comprising amino acid substitutions at one or more supporting (S) residues identified from the "prox" line as shown in FIGS. 3A or 3B; [0572]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0573]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0574]62A. A method for selecting modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0575]a. obtaining an array of modified antibody variable domains comprising amino acid substitutions at one or more interfacial (I) residues identified from the "prox" line as shown in FIGS. 3A or 3B; [0576]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0577]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0578]63A. The method of embodiment 59, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0579]64A. The method of embodiment 63, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0580]65A. The method of embodiment 59, wherein the contacting residue is in CDR2 in a light chain variable domain.

[0581]66A. The method of embodiment 65, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0582]67A. The method of embodiment 59, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0583]68A. The method of embodiment 67, wherein the contacting residue is at position 32 or 33 in CDR1.

[0584]69A. The method of embodiment 59, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0585]70A. The method of embodiment 69, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0586]71A. The method of any one of embodiments 59 to 62, wherein the amino acid substitutions are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0587]72A. The method of any one of embodiments 59 to 62, wherein the amino acid substitutions are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0588]73A. The method of embodiment 72, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0589]74A. The method of embodiment 72, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0590]75A. The method of any one of embodiments 59 to 62, wherein the variable domain is from a humanized antibody.

[0591]76A. The method of any one of embodiments 59 to 62, wherein the variable domain is from a human antibody.

[0592]77A. The method of any one of embodiments 59 to 62, wherein binding affinity is determined by measuring K_off.

[0593]78A. A method for generating an array of modified antibody variable domains with eighteen amino acid changes at one or more contacting (C) residues from a collection of modified variable domains, said method comprising: [0594]a. obtaining a collection of modified antibody variable domains containing amino acid changes at one or more contacting (C) residues; [0595]b. sequencing the collection of modified variable domains; and [0596]c. arranging each sequenced modified antibody variable domain comprising one of the eighteen amino acid changes at one or more contacting (C) residue to generate an array of modified variable domains with eighteen amino acid changes at one or more contacting (C) residues.

[0597]79A. The method of embodiment 78, wherein the collection is a library.

[0598]80A. A method for generating an array of modified variable domains with eighteen amino acid changes at one or more contacting (C) residues, said method comprising: [0599]a. synthesizing polynucleotides that encode sequences that vary at one or more contacting (C) residues and contain eighteen amino acid changes at each contacting (C) residue to generate modified antibody variable domains; and [0600]b. arranging each synthesized polynucleotide from step (a) to generate an array of synthesized polynucleotides with eighteen amino acid changes at one or more contacting (C) residues.

[0601]81A. A method for generating an array of modified variable domains with eighteen amino acid changes at one or more contacting (C) residues, said method comprising: [0602]a. synthesizing polynucleotides that encode sequences that vary at one or more contacting (C) residues and contain eighteen amino acid changes at each contacting (C) residue to generate modified antibody variable domains; [0603]b. transfecting each synthesized polynucleotide of step (a) separately into a host cell to generate clones comprising the synthesized polynucleotides; and [0604]c. arranging each clone from step (b) to generate an array of clones capable of expressing modified variable domains with eighteen amino acid changes at one or more contacting (C) residues.

[0605]82A. The method of any one of embodiments 78, 80 or 81, wherein each contacting (C) residue in the antibody variable domain is separately changed.

[0606]83A. The method of any one of embodiments 78, 80 or 81, wherein one or more contacting (C) residues in the antibody variable domain are simultaneously changed.

[0607]84A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is in complementarity determining domain-1 (CDR1) in a light chain variable domain.

[0608]85A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is at position 28, 30 or 31 in CDR1.

[0609]86A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is in CDR2 in a light chain variable domain.

[0610]87A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is at position 50, 51 or 53 in CDR2.

[0611]88A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is in CDR1 in a heavy chain variable domain.

[0612]89A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is at position 32 or 33 in CDR1.

[0613]90A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is in CDR2 in a heavy chain variable domain.

[0614]91A. The method of any one of embodiments 78, 80 or 81, wherein the contacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

[0615]92A. The method of any one of any one of embodiments 78, 80 or 81, wherein the amino acid changes are alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

[0616]93A. The method of any one of embodiments 78, 80 or 81, wherein the amino acid changes are introduced by PCR mutagenesis using primers which comprise one of seven degenerate codons.

[0617]94A. The method of any one of embodiments 78, 80 or 81, wherein the degenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

[0618]95A. The method of any one of embodiments 78, 80 or 81, wherein the degenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

[0619]96A. The method of any one of embodiments 78, 80 or 81, wherein the variable domain is from a humanized antibody.

[0620]97A. The method of any one of embodiments 78, 80 or 81, wherein the variable domain is from a human antibody.

[0621]98A. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain modified variable domains with enhanced binding affinity to the binding partner, the method comprising: [0622]a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A or 3B; [0623]b. substituting one or more contacting (C), peripheral (P), supporting (S) and interfacial (I) amino acid residues in the antibody variable domain with other amino acid residues to generate an array of modified variable domains; [0624]c. screening the array of modified variable domains for binding affinity to the binding partner; and [0625]d. obtaining modified variable domains with enhanced binding affinity to the binding partner.

[0626]99A. The method of embodiment 98, wherein each contacting (C), peripheral (P), supporting (S) and interfacial (I) residue in the antibody variable domain is separately substituted.

[0627]100A. A method of making modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0628]a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode contacting (C), peripheral (P), supporting (S) and interfacial (I) residues identified from the "prox" line as shown in FIGS. 3A or 3B to produce amino acid substitutions at C residues to generate an array of modified antibody variable domains; and [0629]b. selecting modified variable domains from the array that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0630]101A. The method of embodiment100, wherein each contacting (C), peripheral (P), supporting (S) and interfacial (I) residue in the antibody variable domain is separately substituted.

[0631]102A. A method for selecting modified variable domains of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising: [0632]a. obtaining an array of modified antibody variable domains comprising amino acid substitutions at one or more contacting (C), peripheral (P), supporting (S) and interfacial (I) residue identified from the "prox" line as shown in FIGS. 3A or 3B; [0633]b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; and [0634]c. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

[0635]103A. The method of embodiment 102, wherein each contacting (C), peripheral (P), supporting (S) and interfacial (I) residue in the antibody variable domain is separately substituted.

[0636]1B. An antibody comprising an ING-1 heavy chain variable region as set forth in SEQ ID NO: 579 comprising a substitution at position 28 or 30 in HCDR1.

[0637]2B. The heavy chain variable region of embodiment 1B, wherein the substitution at position 28 is selected from the group consisting of: T28V, T28I and T28P.

[0638]3B. The heavy chain variable region of embodiment 1B, wherein the substitution at position 30 is T30Y.

[0639]4B. An antibody comprising an ING-1 heavy chain variable region as set forth in SEQ ID NO: 579 comprising a substitution at position 59 in HCDR2.

[0640]5B. The heavy chain variable region of embodiment 4B, wherein the substitution at position 59 is T59W.

[0641]6B. An antibody comprising an ING-1 heavy chain variable region as set forth in SEQ ID NO: 579 comprising a substitution at position 100, 101 or 102 in HCDR3.

[0642]7B. The heavy chain variable region of embodiment 6B, wherein the substitution at position 100 is G100R.

[0643]8B. The heavy chain variable region of embodiment 6B, wherein the substitution at position 101 is selected from the group consisting of: S101K, S101Q, S101V, S101I, S101G.

[0644]9B. The heavy chain variable region of embodiment 6B, wherein the substitution at position 102 in HCDR3 is selected from the group consisting of: A102R, A102H, A102Y, A102W, A102F and A102G.

[0645]10B. An antibody comprising an ING-1 light chain variable region as set forth in SEQ ID NO: 580 comprising a substitution at position 28 or 29 in LCDR1.

[0646]11B. The light chain variable region of embodiment 10B, wherein the substitution at position 28 in LCDR1 is selected from the group consisting of: S28R, S28K, S28H, S28Y, S28F, S28Q, S28V, S28I and S28L.

[0647]12B. The light chain variable region of embodiment 10B, wherein the substitution at position 29 in LCDR1 is selected from the group consisting of L29S and L29A.

[0648]13B. An antibody comprising an ING-1 light chain variable region as set forth in SEQ ID NO: 580 comprising a substitution at 54, 55 or 58 in LCDR2.

[0649]14B. The light chain variable region of embodiment 13B, wherein the substitution at position 54 in LCDR2 is selected from the group consisting of: Y54K and Y54L.

[0650]15B. The light chain variable region of embodiment 13B, wherein the substitution at position 55 in LCDR2 is selected from the group consisting of: Q55R, Q55H and Q55W.

[0651]16B. The light chain variable region of embodiment 13B, wherein the substitution at position 58 in LCDR2 is selected from the group consisting of: N58W, N58V, N58I and N58P.

[0652]17B. An antibody comprising an ING-1 light chain variable region as set forth in SEQ ID NO: 580 comprising a substitution at position 97, 98, 99 or 100 in LCDR3.

[0653]18B. The light chain variable region of embodiment 17B, wherein the substitution at position 97 in LCDR3 is L97I.

[0654]19B. The light chain variable region of embodiment 17B, wherein the substitution at position 98 in LCDR3 is selected from the group consisting of: E98R, E98K, E98T, E98S and E98L.

[0655]20B. The light chain variable region of embodiment 17B, wherein the substitution at position 99 in LCDR3 is L99I.

[0656]21B. The light chain variable region of embodiment 17B, wherein the substitution at position 100 in LCDR3 is P100Y.

[0657]22B. An antibody comprising an XPA-23 light chain variable region as set forth in SEQ ID NO: 582 comprising a substitution at position 27, 28 or 30 in LCDR1.

[0658]23B. The light chain variable region of embodiment 22B, wherein the substitution at position 27 in LCDR1 is selected from the group consisting of: Q27S, Q27F and Q27G.

[0659]24B. The light chain variable region of embodiment 22B, wherein the substitution at position 28 in LCDR1 is selected from the group consisting of: D28I, D28S and D28W.

[0660]25B. The light chain variable region of embodiment 22B, wherein the substitution at position 30 in LCDR1 is N30F.

[0661]26B. An antibody comprising an XPA-23 light chain variable region as set forth in SEQ ID NO: 582 comprising a substitution at position 51 or 53 in LCDR2.

[0662]27B. The light chain variable region of embodiment 26B, wherein the substitution at position 51 in LCDR2 is A51 G.

[0663]28B. The light chain variable region of embodiment 26B, wherein the substitution at position 53 in LCDR2 is selected from the group consisting of: S53K and S53R.

[0664]29B. An antibody comprising an XPA-23 light chain variable region as set forth in SEQ ID NO: 582 comprising a substitution at position 92, 93, 95 or 96 in LCDR3.

[0665]30B. The light chain variable region of embodiment 29B, wherein the substitution at position 92 in LCDR3 is D92S.

[0666]31B. The light chain variable region of embodiment 29B, wherein the substitution at position 93 in LCDR3 is selected from the group consisting of: S93D and S93E.

[0667]32B. The light chain variable region of embodiment 29B, wherein the substitution at position 95 in LCDR3 is selected from the group consisting of: P95S and P95A.

[0668]33B. The light chain variable region of embodiment 29B, wherein the substitution at position 96 in LCDR3 is L96W.

[0669]34B. An antibody comprising an XPA-23 heavy chain variable region as set forth in SEQ ID NO: 581 comprising a substitution at position 135, 138, 139, 140 or 142 in HCDR1.

[0670]35B. The heavy chain variable region of embodiment 34B, wherein the substitution at position 135 in HCDR1 is selected from the group consisting of: T135K and T135E.

[0671]36B. The heavy chain variable region of embodiment 34B, wherein the substitution at position 138 in HCDR1 is selected from the group consisting of: K138Y, K138W, K138E, K138L, K138P and K138H.

[0672]37B. The heavy chain variable region of embodiment 34B, wherein the substitution at position 139 in HCDR1 is Y139H.

[0673]38B. The heavy chain variable region of embodiment 34B, wherein the substitution at position 140 in HCDR1 is F140I.

[0674]39B. The heavy chain variable region of embodiment 34B, wherein the substitution at position 142 in HCDR1 is selected from the group consisting of: F142T and F142A.

[0675]40B. An antibody comprising an XPA-23 heavy chain variable region as set forth in SEQ ID NO: 581 comprising a substitution at position 161 or 163 in HCDR2.

[0676]41B. The heavy chain variable region of embodiment 40B, wherein the substitution at position 161 in HCDR2 is selected from the group consisting of: S161R and S161K.

[0677]42B. The heavy chain variable region of embodiment 40B, wherein the substitution at position 163 in HCDR2 is selected from the group consisting of: G163L, G163Q, G163W, G163Y, G163I, G163K, G163R and G163F.

[0678]43B. An antibody comprising an XPA-23 heavy chain variable region as set forth in SEQ ID NO: 581 comprising a substitution at position 208, 210, 211 or 212 in HCDR3.

[0679]44B. The heavy chain variable region of embodiment 43B, wherein the substitution at position 208 in HCDR3 is Y208L.

[0680]45B. The heavy chain variable region of embodiment 43B, wherein the substitution at position 210 in HCDR3 is G210V.

[0681]46B. The heavy chain variable region of embodiment 43B, wherein the substitution at position 211 in HCDR3 is selected from the group consisting of: N211A and N211V.

[0682]47B. The heavy chain variable region of embodiment 43B, wherein the substitution at position 212 in HCDR3 is selected from the group consisting of: S212E and S212P.

[0683]While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

Sequence CWU 1

981120DNAhuman 1actagagcgg caggagatgg 20255DNAArtificial SequenceSynthesized L1-1B primer 2ccatctcctg ccgctctagt argagtctcc tacatagtaa tggcatcact tattt 55355DNAArtificial SequenceSynthesized L1-1P1 (ING-1) primer 3ccatctcctg ccgctctagt wmcagtctcc tacatagtaa tggcatcact tattt 55455DNAArtificial SequenceSynthesized L1-1P2 (ING-1) primer 4ccatctcctg ccgctctagt casagtctcc tacatagtaa tggcatcact tattt 55555DNAArtificial SequenceSynthesized L1-1A (ING-1) primer 5ccatctcctg ccgctctagt gasagtctcc tacatagtaa tggcatcact tattt 55655DNAArtificial SequenceSynthesized L1-1NP1 (ING-1) primer 6ccatctcctg ccgctctagt ntcagtctcc tacatagtaa tggcatcact tattt 55755DNAArtificial SequenceSynthesized L1-1NP2 (ING-1) primer 7ccatctcctg ccgctctagt kggagtctcc tacatagtaa tggcatcact tattt 55855DNAArtificial SequenceSynthesized L1-1NP3 primer 8ccatctcctg ccgctctagt scgagtctcc tacatagtaa tggcatcact tattt 55955DNAArtificial SequenceSynthesized L1-2B (ING-1) primer 9ccatctcctg ccgctctagt aagargctcc tacatagtaa tggcatcact tattt 551055DNAArtificial SequenceSynthesized L1-2P1 (ING-1) primer 10ccatctcctg ccgctctagt aagwmcctcc tacatagtaa tggcatcact tattt 551155DNAArtificial SequenceSynthesized L1-2P2 primer 11ccatctcctg ccgctctagt aagcasctcc tacatagtaa tggcatcact tattt 551255DNAArtificial SequenceSynthesized L1-2A primer 12ccatctcctg ccgctctagt aaggasctcc tacatagtaa tggcatcact tattt 551355DNAArtificial SequenceSynthesized L1-2NP1 (ING-1) primer 13ccatctcctg ccgctctagt aagntcctcc tacatagtaa tggcatcact tattt 551455DNAArtificial SequenceSynthesized L1-2NP2 (ING-1) primer 14ccatctcctg ccgctctagt aagkggctcc tacatagtaa tggcatcact tattt 551555DNAArtificial SequenceSynthesized L1-2NP3 (ING-1) primer 15ccatctcctg ccgctctagt aagscgctcc tacatagtaa tggcatcact tattt 551655DNAArtificial SequenceSynthesized L1-3B (ING-1) primer 16ccatctcctg ccgctctagt aagagtargc tacatagtaa tggcatcact tattt 551755DNAArtificial SequenceSynthesized L1-3P1 (ING-1) primer 17ccatctcctg ccgctctagt aagagtwmcc tacatagtaa tggcatcact tattt 551855DNAArtificial SequenceSynthesized L1-3P2 (ING-1) primer 18ccatctcctg ccgctctagt aagagtcasc tacatagtaa tggcatcact tattt 551955DNAArtificial SequenceSynthesized L1-3A (ING-1) primer 19ccatctcctg ccgctctagt aagagtgasc tacatagtaa tggcatcact tattt 552055DNAArtificial SequenceSynthesized L1-3NP1 (ING-1) primer 20ccatctcctg ccgctctagt aagagtntcc tacatagtaa tggcatcact tattt 552155DNAArtificial SequenceSynthesized L1-3NP2 (ING-1) primer 21ccatctcctg ccgctctagt aagagtkggc tacatagtaa tggcatcact tattt 552255DNAArtificial SequenceSynthesized L1-3NP3 (ING-1) primer 22ccatctcctg ccgctctagt aagagtscgc tacatagtaa tggcatcact tattt 552355DNAArtificial SequenceSynthesized L1-4B (ING-1) primer 23ccatctcctg ccgctctagt aagagtctca rgcatagtaa tggcatcact tattt 552455DNAArtificial SequenceSynthesized L1-4P1 (ING-1) primer 24ccatctcctg ccgctctagt aagagtctcw mccatagtaa tggcatcact tattt 552555DNAArtificial SequenceSynthesized L1-4P2 (ING-1) primer 25ccatctcctg ccgctctagt aagagtctcc ascatagtaa tggcatcact tattt 552655DNAArtificial SequenceSynthesized L1-4A primer 26ccatctcctg ccgctctagt aagagtctcg ascatagtaa tggcatcact tattt 552755DNAArtificial SequenceSynthesized L1-4NP1 (ING-1) primer 27ccatctcctg ccgctctagt aagagtctcn tccatagtaa tggcatcact tattt 552855DNAArtificial SequenceSynthesized L1-4NP2 (ING-1) primer 28ccatctcctg ccgctctagt aagagtctck ggcatagtaa tggcatcact tattt 552955DNAArtificial SequenceSynthesized L1-4NP3 (ING-1) primer 29ccatctcctg ccgctctagt aagagtctcs cgcatagtaa tggcatcact tattt 553021DNAArtificial SequenceSynthesized L12-R (ING-1) primer 30taggagactc ttactagagc g 213154DNAArtificial SequenceSynthesized L12-1B (ING-1) primer 31cgctctagta agagtctcct aargagtaat ggcatcactt atttgtattg gtat 543254DNAArtificial SequenceSynthesized L12-1P1 (ING-1) primer 32cgctctagta agagtctcct awmcagtaat ggcatcactt atttgtattg gtat 543354DNAArtificial SequenceSynthesized L12-1P2 (ING-1) primer 33cgctctagta agagtctcct acasagtaat ggcatcactt atttgtattg gtat 543454DNAArtificial SequenceSynthesized L12-1A (ING-1) primer 34cgctctagta agagtctcct agasagtaat ggcatcactt atttgtattg gtat 543554DNAArtificial SequenceSynthesized L12-1NP1 (ING-1) primer 35cgctctagta agagtctcct antcagtaat ggcatcactt atttgtattg gtat 543654DNAArtificial SequenceSynthesized L12-1NP2 (ING-1) primer 36cgctctagta agagtctcct akggagtaat ggcatcactt atttgtattg gtat 543754DNAArtificial SequenceSynthesized L12-1NP3 (ING-1) primer 37cgctctagta agagtctcct ascgagtaat ggcatcactt atttgtattg gtat 543854DNAArtificial SequenceSynthesized L12-2B (ING-1) primer 38cgctctagta agagtctcct acatargaat ggcatcactt atttgtattg gtat 543954DNAArtificial SequenceSynthesized L12-2P1 (ING-1) primer 39cgctctagta agagtctcct acatwmcaat ggcatcactt atttgtattg gtat 544054DNAArtificial SequenceSynthesized L12-2P2 (ING-1) primer 40cgctctagta agagtctcct acatcasaat ggcatcactt atttgtattg gtat 544154DNAArtificial SequenceSynthesized L12-2A (ING-1) primer 41cgctctagta agagtctcct acatgasaat ggcatcactt atttgtattg gtat 544254DNAArtificial SequenceSynthesized L12-2NP1 (ING-1) primer 42cgctctagta agagtctcct acatntcaat ggcatcactt atttgtattg gtat 544354DNAArtificial SequenceSynthesized L12-2NP2 (ING-1) primer 43cgctctagta agagtctcct acatkggaat ggcatcactt atttgtattg gtat 544454DNAArtificial SequenceSynthesized L12-2NP3 (ING-1) primer 44cgctctagta agagtctcct acatscgaat ggcatcactt atttgtattg gtat 544554DNAArtificial SequenceSynthesized L12-3B (ING-1) primer 45cgctctagta agagtctcct acatagtarg ggcatcactt atttgtattg gtat 544654DNAArtificial SequenceSynthesized L12-3P1 (ING-1) primer 46cgctctagta agagtctcct acatagtwmc ggcatcactt atttgtattg gtat 544754DNAArtificial SequenceSynthesized L12-3P2 (ING-1) primer 47cgctctagta agagtctcct acatagtcas ggcatcactt atttgtattg gtat 544854DNAArtificial SequenceSynthesized L12-3A (ING-1) primer 48cgctctagta agagtctcct acatagtgas ggcatcactt atttgtattg gtat 544954DNAArtificial SequenceSynthesized L12-3NP1 (ING-1) primer 49cgctctagta agagtctcct acatagtntc ggcatcactt atttgtattg gtat 545054DNAArtificial SequenceSynthesized L12-3NP2 (ING-1) primer 50cgctctagta agagtctcct acatagtkgg ggcatcactt atttgtattg gtat 545154DNAArtificial SequenceSynthesized L12-3NP3 (ING-1) primer 51cgctctagta agagtctcct acatagtscg ggcatcactt atttgtattg gtat 545222DNAArtificial SequenceSynthesized L13-R (ING-1) primer 52gccattacta tgtaggagac tc 225354DNAArtificial SequenceSynthesized L13-1B (ING-1) primer 53gagtctccta catagtaatg gcargactta tttgtattgg tatttacaga agcc 545454DNAArtificial SequenceSynthesized L13-1P1 (ING-1) primer 54gagtctccta catagtaatg gcwmcactta tttgtattgg tatttacaga agcc 545554DNAArtificial SequenceSynthesized L13-1P2 (ING-1) primer 55gagtctccta catagtaatg gccasactta tttgtattgg tatttacaga agcc 545654DNAArtificial SequenceSynthesized L13-1A (ING-1) primer 56gagtctccta catagtaatg gcgasactta tttgtattgg tatttacaga agcc 545754DNAArtificial SequenceSynthesized L13-1NP1 (ING-1) primer 57gagtctccta catagtaatg gcntcactta tttgtattgg tatttacaga agcc 545854DNAArtificial SequenceSynthesized L13-1NP2 (ING-1) primer 58gagtctccta catagtaatg gckggactta tttgtattgg tatttacaga agcc 545954DNAArtificial SequenceSynthesized L13-1NP3 (ING-1) primer 59gagtctccta catagtaatg gcscgactta tttgtattgg tatttacaga agcc 546054DNAArtificial SequenceSynthesized L13-2B (ING-1) primer 60gagtctccta catagtaatg gcatcargta tttgtattgg tatttacaga agcc 546154DNAArtificial SequenceSynthesized L13-2P1 (ING-1) primer 61gagtctccta catagtaatg gcatcwmcta tttgtattgg tatttacaga agcc 546254DNAArtificial SequenceSynthesized L13-2P2 (ING-1) primer 62gagtctccta catagtaatg gcatccasta tttgtattgg tatttacaga agcc 546354DNAArtificial SequenceSynthesized L13-2A (ING-1) primer 63gagtctccta catagtaatg gcatcgasta tttgtattgg tatttacaga agcc 546454DNAArtificial SequenceSynthesized L13-2NP1 (ING-1) primer 64gagtctccta catagtaatg gcatcntcta tttgtattgg tatttacaga agcc 546554DNAArtificial SequenceSynthesized L13-2NP2 (ING-1) primer 65gagtctccta catagtaatg gcatckggta tttgtattgg tatttacaga agcc 546654DNAArtificial SequenceSynthesized L13-2NP3 (ING-1) primer 66gagtctccta catagtaatg gcatcscgta tttgtattgg tatttacaga agcc 546754DNAArtificial SequenceSynthesized L13-3B (ING-1) primer 67gagtctccta catagtaatg gcatcactar gttgtattgg tatttacaga agcc 546854DNAArtificial SequenceSynthesized L13-3P1 (ING-1) primer 68gagtctccta catagtaatg gcatcactwm cttgtattgg tatttacaga agcc 546954DNAArtificial SequenceSynthesized L13-3P2 (ING-1) primer 69gagtctccta catagtaatg gcatcactca sttgtattgg tatttacaga agcc 547054DNAArtificial SequenceSynthesized L13-3A (ING-1) primer 70gagtctccta catagtaatg gcatcactga sttgtattgg tatttacaga agcc 547154DNAArtificial SequenceSynthesized L13-3NP1 (ING-1) primer 71gagtctccta catagtaatg gcatcactnt cttgtattgg tatttacaga agcc 547254DNAArtificial SequenceSynthesized L13-3NP2 (ING-1) primer 72gagtctccta catagtaatg gcatcactkg gttgtattgg tatttacaga agcc 547354DNAArtificial SequenceSynthesized L13-3NP3 (ING-1) primer 73gagtctccta catagtaatg gcatcactsc gttgtattgg tatttacaga agcc 547421DNAArtificial SequenceSynthesized L2R (ING-1) primer 74aatcaggagc tgaggagact g 217556DNAArtificial SequenceSynthesized L2-1B (ING-1) primer 75cagtctcctc agctcctgat targcagatg tccaaccttg cctcaggagt cccaga 567656DNAArtificial SequenceSynthesized L2-1P1 (ING-1) primer 76cagtctcctc agctcctgat twmccagatg tccaaccttg cctcaggagt cccaga 567756DNAArtificial SequenceSynthesized L2-1P2 (ING-1) primer 77cagtctcctc agctcctgat tcascagatg tccaaccttg cctcaggagt cccaga 567856DNAArtificial SequenceSynthesized L2-1A (ING-1) primer 78cagtctcctc agctcctgat tgascagatg tccaaccttg cctcaggagt cccaga 567956DNAArtificial SequenceSynthesized L2-1NP1 (ING-1) primer 79cagtctcctc agctcctgat tntccagatg tccaaccttg cctcaggagt cccaga 568056DNAArtificial SequenceSynthesized L2-1NP2 (ING-1) primer 80cagtctcctc agctcctgat tkggcagatg tccaaccttg cctcaggagt cccaga 568156DNAArtificial SequenceSynthesized L2-1NP3 (ING-1) primer 81cagtctcctc agctcctgat tscgcagatg tccaaccttg cctcaggagt cccaga 568256DNAArtificial SequenceSynthesized L2-2B (ING-1) primer 82cagtctcctc agctcctgat ttatargatg tccaaccttg cctcaggagt cccaga 568356DNAArtificial SequenceSynthesized L2-2P1 (ING-1) primer 83cagtctcctc agctcctgat ttatwmcatg tccaaccttg cctcaggagt cccaga 568456DNAArtificial SequenceSynthesized L2-2P2 (ING-1) primer 84cagtctcctc agctcctgat ttatcasatg tccaaccttg cctcaggagt cccaga 568556DNAArtificial SequenceSynthesized L2-2A (ING-1) primer 85cagtctcctc agctcctgat ttatgasatg tccaaccttg cctcaggagt cccaga 568656DNAArtificial SequenceSynthesized L2-2NP1 (ING-1) primer 86cagtctcctc agctcctgat ttatntcatg tccaaccttg cctcaggagt cccaga 568756DNAArtificial SequenceSynthesized L2-2NP2 (ING-1) primer 87cagtctcctc agctcctgat ttatkggatg tccaaccttg cctcaggagt cccaga 568856DNAArtificial SequenceSynthesized L2-2NP3 (ING-1) primer 88cagtctcctc agctcctgat ttatscgatg tccaaccttg cctcaggagt cccaga 568956DNAArtificial SequenceSynthesized L2-3B (ING-1) primer 89cagtctcctc agctcctgat ttatcagarg tccaaccttg cctcaggagt cccaga 569056DNAArtificial SequenceSynthesized L2-3P1 (ING-1) primer 90cagtctcctc agctcctgat ttatcagwmc tccaaccttg cctcaggagt cccaga 569156DNAArtificial SequenceSynthesized L2-3P2 (ING-1) primer 91cagtctcctc agctcctgat ttatcagcas tccaaccttg cctcaggagt cccaga 569256DNAArtificial SequenceSynthesized L2-3A (ING-1) primer 92cagtctcctc agctcctgat ttatcaggas tccaaccttg cctcaggagt cccaga 569356DNAArtificial SequenceSynthesized L2-3NP1 (ING-1) primer 93cagtctcctc agctcctgat ttatcagntc tccaaccttg cctcaggagt cccaga 569456DNAArtificial SequenceSynthesized L2-3NP2 (ING-1) primer 94cagtctcctc agctcctgat ttatcagkgg tccaaccttg cctcaggagt cccaga 569556DNAArtificial SequenceSynthesized L2-3NP3 (ING-1) primer 95cagtctcctc agctcctgat ttatcagscg tccaaccttg cctcaggagt cccaga 569656DNAArtificial SequenceSynthesized L2-4B (ING-1) primer 96cagtctcctc agctcctgat ttatcagatg argaaccttg cctcaggagt cccaga 569756DNAArtificial SequenceSynthesized L2-4P1 (ING-1) primer 97cagtctcctc agctcctgat ttatcagatg wmcaaccttg cctcaggagt cccaga 569856DNAArtificial SequenceSynthesized L2-4P2 (ING-1) primer 98cagtctcctc agctcctgat ttatcagatg casaaccttg cctcaggagt cccaga 569956DNAArtificial SequenceSynthesized L2-4A (ING-1) primer 99cagtctcctc agctcctgat ttatcagatg gasaaccttg cctcaggagt cccaga 5610056DNAArtificial SequenceSynthesized L2-4NP1 (ING-1) primer 100cagtctcctc agctcctgat ttatcagatg ntcaaccttg cctcaggagt cccaga 5610156DNAArtificial SequenceSynthesized L2-4NP2 (ING-1) primer 101cagtctcctc agctcctgat ttatcagatg kggaaccttg cctcaggagt cccaga 5610256DNAArtificial SequenceSynthesized L2-4NP3 (ING-1) primer 102cagtctcctc agctcctgat ttatcagatg scgaaccttg cctcaggagt cccaga 5610356DNAArtificial SequenceSynthesized L2-5B (ING-1) primer 103cagtctcctc agctcctgat ttatcagatg tccargcttg cctcaggagt cccaga 5610456DNAArtificial SequenceSynthesized L2-5P1 (ING-1) primer 104cagtctcctc agctcctgat ttatcagatg tccwmccttg cctcaggagt cccaga 5610556DNAArtificial SequenceSynthesized L2-5P2 (ING-1) primer 105cagtctcctc agctcctgat ttatcagatg tcccascttg cctcaggagt cccaga 5610656DNAArtificial SequenceSynthesized L2-5A (ING-1) primer 106cagtctcctc agctcctgat ttatcagatg tccgascttg cctcaggagt cccaga 5610756DNAArtificial SequenceSynthesized L2-5NP1 (ING-1) primer 107cagtctcctc agctcctgat ttatcagatg tccntccttg cctcaggagt cccaga 5610856DNAArtificial SequenceSynthesized L2-5NP2 (ING-1) primer 108cagtctcctc agctcctgat ttatcagatg tcckggcttg cctcaggagt cccaga 5610956DNAArtificial SequenceSynthesized L2-5NP3 (ING-1) primer 109cagtctcctc agctcctgat ttatcagatg tccscgcttg cctcaggagt cccaga 5611024DNAArtificial SequenceSynthesized L3R (ING-1) primer 110attttgagca cagtaataaa cacc 2411159DNAArtificial SequenceSynthesized L3-1B (ING-1) primer 111ggtgtttatt actgtgctca aaatarggaa cttcctcgga cgttcggtgg aggcaccaa 5911259DNAArtificial SequenceSynthesized L3-1P1 (ING-1) primer 112ggtgtttatt actgtgctca aaatwmcgaa cttcctcgga cgttcggtgg aggcaccaa 5911359DNAArtificial SequenceSynthesized L3-1P2 (ING-1) primer 113ggtgtttatt actgtgctca aaatcasgaa cttcctcgga cgttcggtgg aggcaccaa 5911459DNAArtificial SequenceSynthesized L3-1A (ING-1) primer 114ggtgtttatt actgtgctca aaatgasgaa cttcctcgga cgttcggtgg aggcaccaa 5911559DNAArtificial SequenceSynthesized L3-1NP1 (ING-1) primer 115ggtgtttatt actgtgctca aaatntcgaa cttcctcgga cgttcggtgg aggcaccaa 5911659DNAArtificial SequenceSynthesized L3-1NP2 (ING-1) primer 116ggtgtttatt actgtgctca aaatkgggaa cttcctcgga cgttcggtgg aggcaccaa 5911759DNAArtificial SequenceSynthesized L3-1NP3 (ING-1) primer 117ggtgtttatt actgtgctca aaatscggaa cttcctcgga cgttcggtgg aggcaccaa 5911859DNAArtificial SequenceSynthesized L3-2B (ING-1) primer 118ggtgtttatt actgtgctca aaatctaarg cttcctcgga cgttcggtgg aggcaccaa 5911959DNAArtificial SequenceSynthesized L3-2P1 (ING-1) primer 119ggtgtttatt actgtgctca aaatctawmc cttcctcgga cgttcggtgg aggcaccaa 5912059DNAArtificial SequenceSynthesized L3-2P2 (ING-1) primer 120ggtgtttatt actgtgctca aaatctacas cttcctcgga cgttcggtgg aggcaccaa 5912159DNAArtificial SequenceSynthesized L3-2A (ING-1) primer 121ggtgtttatt actgtgctca aaatctagas cttcctcgga cgttcggtgg aggcaccaa 5912259DNAArtificial SequenceSynthesized L3-2NP1 (ING-1) primer 122ggtgtttatt actgtgctca aaatctantc cttcctcgga cgttcggtgg aggcaccaa 5912359DNAArtificial SequenceSynthesized L3-2NP2 (ING-1) primer 123ggtgtttatt actgtgctca aaatctakgg cttcctcgga cgttcggtgg aggcaccaa 5912459DNAArtificial SequenceSynthesized L3-2NP3 (ING-1) primer 124ggtgtttatt actgtgctca aaatctascg cttcctcgga cgttcggtgg aggcaccaa 5912559DNAArtificial SequenceSynthesized L3-3B (ING-1) primer 125ggtgtttatt actgtgctca aaatctagaa argcctcgga cgttcggtgg aggcaccaa 5912659DNAArtificial SequenceSynthesized L3-3P1 (ING-1) primer 126ggtgtttatt actgtgctca aaatctagaa wmccctcgga cgttcggtgg aggcaccaa

5912759DNAArtificial SequenceSynthesized L3-3P2 (ING-1) primer 127ggtgtttatt actgtgctca aaatctagaa cascctcgga cgttcggtgg aggcaccaa 5912859DNAArtificial SequenceSynthesized L3-3A (ING-1) primer 128ggtgtttatt actgtgctca aaatctagaa gascctcgga cgttcggtgg aggcaccaa 5912959DNAArtificial SequenceSynthesized L3-3NP1 (ING-1) primer 129ggtgtttatt actgtgctca aaatctagaa ntccctcgga cgttcggtgg aggcaccaa 5913059DNAArtificial SequenceSynthesized L3-3NP2 (ING-1) primer 130ggtgtttatt actgtgctca aaatctagaa kggcctcgga cgttcggtgg aggcaccaa 5913159DNAArtificial SequenceSynthesized L3-3NP3 (ING-1) primer 131ggtgtttatt actgtgctca aaatctagaa scgcctcgga cgttcggtgg aggcaccaa 5913259DNAArtificial SequenceSynthesized L3-4B (ING-1) primer 132ggtgtttatt actgtgctca aaatctagaa cttargcgga cgttcggtgg aggcaccaa 5913359DNAArtificial SequenceSynthesized L3-4P1 (ING-1) primer 133ggtgtttatt actgtgctca aaatctagaa cttwmccgga cgttcggtgg aggcaccaa 5913459DNAArtificial SequenceSynthesized L3-4P2 (ING-1) primer 134ggtgtttatt actgtgctca aaatctagaa cttcascgga cgttcggtgg aggcaccaa 5913559DNAArtificial SequenceSynthesized L3-4A (ING-1) primer 135ggtgtttatt actgtgctca aaatctagaa cttgascgga cgttcggtgg aggcaccaa 5913659DNAArtificial SequenceSynthesized L3-4NP1 (ING-1) primer 136ggtgtttatt actgtgctca aaatctagaa cttntccgga cgttcggtgg aggcaccaa 5913759DNAArtificial SequenceSynthesized L3-4NP2 (ING-1) primer 137ggtgtttatt actgtgctca aaatctagaa cttkggcgga cgttcggtgg aggcaccaa 5913859DNAArtificial SequenceSynthesized L3-4NP3 (ING-1) primer 138ggtgtttatt actgtgctca aaatctagaa cttscgcgga cgttcggtgg aggcaccaa 5913959DNAArtificial SequenceSynthesized L3-5B (ING-1) primer 139ggtgtttatt actgtgctca aaatctagaa cttcctarga cgttcggtgg aggcaccaa 5914059DNAArtificial SequenceSynthesized L3-5P1 (ING-1) primer 140ggtgtttatt actgtgctca aaatctagaa cttcctwmca cgttcggtgg aggcaccaa 5914159DNAArtificial SequenceSynthesized L3-5P2 (ING-1) primer 141ggtgtttatt actgtgctca aaatctagaa cttcctcasa cgttcggtgg aggcaccaa 5914259DNAArtificial SequenceSynthesized L3-5A (ING-1) primer 142ggtgtttatt actgtgctca aaatctagaa cttcctgasa cgttcggtgg aggcaccaa 5914359DNAArtificial SequenceSynthesized L3-5NP1 (ING-1) primer 143ggtgtttatt actgtgctca aaatctagaa cttcctntca cgttcggtgg aggcaccaa 5914459DNAArtificial SequenceSynthesized L3-5NP2 (ING-1) primer 144ggtgtttatt actgtgctca aaatctagaa cttcctkgga cgttcggtgg aggcaccaa 5914559DNAArtificial SequenceSynthesized L3-5NP3 (ING-1) primer 145ggtgtttatt actgtgctca aaatctagaa cttcctscga cgttcggtgg aggcaccaa 5914621DNAArtificial SequenceSynthesized H1R (ING-1) primer 146atatccagaa gccttgcagg a 2114759DNAArtificial SequenceSynthesized H1-1B (ING-1) primer 147tcctgcaagg cttctggata targttcaca aaatatggaa tgaactgggt gaagcaggc 5914859DNAArtificial SequenceSynthesized H1-1P1 (ING-1) primer 148tcctgcaagg cttctggata twmcttcaca aaatatggaa tgaactgggt gaagcaggc 5914959DNAArtificial SequenceSynthesized H1-1P2 (ING-1) primer 149tcctgcaagg cttctggata tcasttcaca aaatatggaa tgaactgggt gaagcaggc 5915059DNAArtificial SequenceSynthesized H1-1A (ING-1) primer 150tcctgcaagg cttctggata tgasttcaca aaatatggaa tgaactgggt gaagcaggc 5915159DNAArtificial SequenceSynthesized H1-1NP1 (ING-1) primer 151tcctgcaagg cttctggata tntcttcaca aaatatggaa tgaactgggt gaagcaggc 5915259DNAArtificial SequenceSynthesized H1-1NP2 (ING-1) primer 152tcctgcaagg cttctggata tkggttcaca aaatatggaa tgaactgggt gaagcaggc 5915359DNAArtificial SequenceSynthesized H1-1NP3 (ING-1) primer 153tcctgcaagg cttctggata tscgttcaca aaatatggaa tgaactgggt gaagcaggc 5915459DNAArtificial SequenceSynthesized H1-2B (ING-1) primer 154tcctgcaagg cttctggata taccttcarg aaatatggaa tgaactgggt gaagcaggc 5915559DNAArtificial SequenceSynthesized H1-2P1 (ING-1) primer 155tcctgcaagg cttctggata taccttcwmc aaatatggaa tgaactgggt gaagcaggc 5915659DNAArtificial SequenceSynthesized H1-2P2 (ING-1) primer 156tcctgcaagg cttctggata taccttccas aaatatggaa tgaactgggt gaagcaggc 5915759DNAArtificial SequenceSynthesized H1-2A (ING-1) primer 157tcctgcaagg cttctggata taccttcgas aaatatggaa tgaactgggt gaagcaggc 5915859DNAArtificial SequenceSynthesized H1-2NP1 (ING-1) primer 158tcctgcaagg cttctggata taccttcntc aaatatggaa tgaactgggt gaagcaggc 5915959DNAArtificial SequenceSynthesized H1-2NP2 (ING-1) primer 159tcctgcaagg cttctggata taccttckgg aaatatggaa tgaactgggt gaagcaggc 5916059DNAArtificial SequenceSynthesized H1-2NP3 (ING-1) primer 160tcctgcaagg cttctggata taccttcscg aaatatggaa tgaactgggt gaagcaggc 5916159DNAArtificial SequenceSynthesized H1-3B (ING-1) primer 161tcctgcaagg cttctggata taccttcaca argtatggaa tgaactgggt gaagcaggc 5916259DNAArtificial SequenceSynthesized H1-3P1 (ING-1) primer 162tcctgcaagg cttctggata taccttcaca wmctatggaa tgaactgggt gaagcaggc 5916359DNAArtificial SequenceSynthesized H1-3P2 (ING-1) primer 163tcctgcaagg cttctggata taccttcaca castatggaa tgaactgggt gaagcaggc 5916459DNAArtificial SequenceSynthesized H1-3A (ING-1) primer 164tcctgcaagg cttctggata taccttcaca gastatggaa tgaactgggt gaagcaggc 5916559DNAArtificial SequenceSynthesized H1-3NP1 (ING-1) primer 165tcctgcaagg cttctggata taccttcaca ntctatggaa tgaactgggt gaagcaggc 5916659DNAArtificial SequenceSynthesized H1-3NP2 (ING-1) primer 166tcctgcaagg cttctggata taccttcaca kggtatggaa tgaactgggt gaagcaggc 5916759DNAArtificial SequenceSynthesized H1-3NP3 (ING-1) primer 167tcctgcaagg cttctggata taccttcaca scgtatggaa tgaactgggt gaagcaggc 5916859DNAArtificial SequenceSynthesized H1-4B (ING-1) primer 168tcctgcaagg cttctggata taccttcaca aaaargggaa tgaactgggt gaagcaggc 5916959DNAArtificial SequenceSynthesized H1-4P1 (ING-1) primer 169tcctgcaagg cttctggata taccttcaca aaawmcggaa tgaactgggt gaagcaggc 5917059DNAArtificial SequenceSynthesized H1-4P2 (ING-1) primer 170tcctgcaagg cttctggata taccttcaca aaacasggaa tgaactgggt gaagcaggc 5917159DNAArtificial SequenceSynthesized H1-4A (ING-1) primer 171tcctgcaagg cttctggata taccttcaca aaagasggaa tgaactgggt gaagcaggc 5917259DNAArtificial SequenceSynthesized H1-4NP1 (ING-1) primer 172tcctgcaagg cttctggata taccttcaca aaantcggaa tgaactgggt gaagcaggc 5917359DNAArtificial SequenceSynthesized H1-4NP2 (ING-1) primer 173tcctgcaagg cttctggata taccttcaca aaakggggaa tgaactgggt gaagcaggc 5917459DNAArtificial SequenceSynthesized H1-4NP3 (ING-1) primer 174tcctgcaagg cttctggata taccttcaca aaascgggaa tgaactgggt gaagcaggc 5917559DNAArtificial SequenceSynthesized H1-5B (ING-1) primer 175tcctgcaagg cttctggata taccttcaca aaatatarga tgaactgggt gaagcaggc 5917659DNAArtificial SequenceSynthesized H1-5P1 (ING-1) primer 176tcctgcaagg cttctggata taccttcaca aaatatwmca tgaactgggt gaagcaggc 5917759DNAArtificial SequenceSynthesized H1-5P2 (ING-1) primer 177tcctgcaagg cttctggata taccttcaca aaatatcasa tgaactgggt gaagcaggc 5917859DNAArtificial SequenceSynthesized H1-5A (ING-1) primer 178tcctgcaagg cttctggata taccttcaca aaatatgasa tgaactgggt gaagcaggc 5917959DNAArtificial SequenceSynthesized H1-5NP1 (ING-1) primer 179tcctgcaagg cttctggata taccttcaca aaatatntca tgaactgggt gaagcaggc 5918059DNAArtificial SequenceSynthesized H1-5NP2 (ING-1) primer 180tcctgcaagg cttctggata taccttcaca aaatatkgga tgaactgggt gaagcaggc 5918159DNAArtificial SequenceSynthesized H1-5NP3 (ING-1) primer 181tcctgcaagg cttctggata taccttcaca aaatatscga tgaactgggt gaagcaggc 5918221DNAArtificial SequenceSynthesized H2R (ING-1) primer 182gcccatccac tttaaaccct t 2118358DNAArtificial SequenceSynthesized H2-1B (ING-1) primer 183aagggtttaa agtggatggg cargataaac acctacactg aagagcctac atatggtg 5818458DNAArtificial SequenceSynthesized H2-1P1 (ING-1) primer 184aagggtttaa agtggatggg cwmcataaac acctacactg aagagcctac atatggtg 5818558DNAArtificial SequenceSynthesized H2-1P2 (ING-1) primer 185aagggtttaa agtggatggg ccasataaac acctacactg aagagcctac atatggtg 5818658DNAArtificial SequenceSynthesized H2-1A (ING-1) primer 186aagggtttaa agtggatggg cgasataaac acctacactg aagagcctac atatggtg 5818758DNAArtificial SequenceSynthesized H2-1NP1 (ING-1) primer 187aagggtttaa agtggatggg cntcataaac acctacactg aagagcctac atatggtg 5818858DNAArtificial SequenceSynthesized H2-1NP2 (ING-1) primer 188aagggtttaa agtggatggg ckggataaac acctacactg aagagcctac atatggtg 5818958DNAArtificial SequenceSynthesized H2-1NP3 (ING-1) primer 189aagggtttaa agtggatggg cscgataaac acctacactg aagagcctac atatggtg 5819058DNAArtificial SequenceSynthesized H2-2B (ING-1) primer 190aagggtttaa agtggatggg ctggataarg acctacactg aagagcctac atatggtg 5819158DNAArtificial SequenceSynthesized H2-2P1 (ING-1) primer 191aagggtttaa agtggatggg ctggatawmc acctacactg aagagcctac atatggtg 5819258DNAArtificial SequenceSynthesized H2-2P2 (ING-1) primer 192aagggtttaa agtggatggg ctggatacas acctacactg aagagcctac atatggtg 5819358DNAArtificial SequenceSynthesized H2-2A (ING-1) primer 193aagggtttaa agtggatggg ctggatagas acctacactg aagagcctac atatggtg 5819458DNAArtificial SequenceSynthesized H2-2NP1 (ING-1) primer 194aagggtttaa agtggatggg ctggatantc acctacactg aagagcctac atatggtg 5819558DNAArtificial SequenceSynthesized H2-2NP2 (ING-1) primer 195aagggtttaa agtggatggg ctggatakgg acctacactg aagagcctac atatggtg 5819658DNAArtificial SequenceSynthesized H2-2NP3 (ING-1) primer 196aagggtttaa agtggatggg ctggatascg acctacactg aagagcctac atatggtg 5819758DNAArtificial SequenceSynthesized H2-3B (ING-1) primer 197aagggtttaa agtggatggg ctggataaac argtacactg aagagcctac atatggtg 5819858DNAArtificial SequenceSynthesized H2-3P1 (ING-1) primer 198aagggtttaa agtggatggg ctggataaac wmctacactg aagagcctac atatggtg 5819958DNAArtificial SequenceSynthesized H2-3P2 (ING-1) primer 199aagggtttaa agtggatggg ctggataaac castacactg aagagcctac atatggtg 5820058DNAArtificial SequenceSynthesized H2-3A (ING-1) primer 200aagggtttaa agtggatggg ctggataaac gastacactg aagagcctac atatggtg 5820158DNAArtificial SequenceSynthesized H2-3NP1 (ING-1) primer 201aagggtttaa agtggatggg ctggataaac ntctacactg aagagcctac atatggtg 5820258DNAArtificial SequenceSynthesized H2-3NP2 (ING-1) primer 202aagggtttaa agtggatggg ctggataaac kggtacactg aagagcctac atatggtg 5820358DNAArtificial SequenceSynthesized H2-3NP3 (ING-1) primer 203aagggtttaa agtggatggg ctggataaac scgtacactg aagagcctac atatggtg 5820458DNAArtificial SequenceSynthesized H2-4B (ING-1) primer 204aagggtttaa agtggatggg ctggataaac accargactg aagagcctac atatggtg 5820558DNAArtificial SequenceSynthesized H2-4P1 (ING-1) primer 205aagggtttaa agtggatggg ctggataaac accwmcactg aagagcctac atatggtg 5820658DNAArtificial SequenceSynthesized H2-4P2 (ING-1) primer 206aagggtttaa agtggatggg ctggataaac acccasactg aagagcctac atatggtg 5820758DNAArtificial SequenceSynthesized H2-4A (ING-1) primer 207aagggtttaa agtggatggg ctggataaac accgasactg aagagcctac atatggtg 5820858DNAArtificial SequenceSynthesized H2-4NP1 (ING-1) primer 208aagggtttaa agtggatggg ctggataaac accntcactg aagagcctac atatggtg 5820958DNAArtificial SequenceSynthesized H2-4NP2 (ING-1) primer 209aagggtttaa agtggatggg ctggataaac acckggactg aagagcctac atatggtg 5821058DNAArtificial SequenceSynthesized H2-4NP3 (ING-1) primer 210aagggtttaa agtggatggg ctggataaac accscgactg aagagcctac atatggtg 5821121DNAArtificial SequenceSynthesized H22-R (ING-1) primer 211gtaggtgttt atccagccca t 2121258DNAArtificial SequenceSynthesized H22-1B (ING-1) primer 212atgggctgga taaacaccta carggaagag cctacatatg gtgatgactt caagggac 5821358DNAArtificial SequenceSynthesized H22-1P1 (ING-1) primer 213atgggctgga taaacaccta cwmcgaagag cctacatatg gtgatgactt caagggac 5821458DNAArtificial SequenceSynthesized H22-1P2 (ING-1) primer 214atgggctgga taaacaccta ccasgaagag cctacatatg gtgatgactt caagggac 5821558DNAArtificial SequenceSynthesized H22-1A (ING-1) primer 215atgggctgga taaacaccta cgasgaagag cctacatatg gtgatgactt caagggac 5821658DNAArtificial SequenceSynthesized H22-1NP1 (ING-1) primer 216atgggctgga taaacaccta cntcgaagag cctacatatg gtgatgactt caagggac 5821758DNAArtificial SequenceSynthesized H22-1NP2 (ING-1) primer 217atgggctgga taaacaccta ckgggaagag cctacatatg gtgatgactt caagggac 5821858DNAArtificial SequenceSynthesized H22-1Np3 (ING-1) primer 218atgggctgga taaacaccta cscggaagag cctacatatg gtgatgactt caagggac 5821958DNAArtificial SequenceSynthesized H22-2B (ING-1) primer 219atgggctgga taaacaccta cactarggag cctacatatg gtgatgactt caagggac 5822058DNAArtificial SequenceSynthesized H22-2P1 (ING-1) primer 220atgggctgga taaacaccta cactwmcgag cctacatatg gtgatgactt caagggac 5822158DNAArtificial SequenceSynthesized H22-2P2 (ING-1) primer 221atgggctgga taaacaccta cactcasgag cctacatatg gtgatgactt caagggac 5822258DNAArtificial SequenceSynthesized H22-2A (ING-1) primer 222atgggctgga taaacaccta cactgasgag cctacatatg gtgatgactt caagggac 5822358DNAArtificial SequenceSynthesized H22-2NP1 (ING-1) primer 223atgggctgga taaacaccta cactntcgag cctacatatg gtgatgactt caagggac 5822458DNAArtificial SequenceSynthesized H22-2NP2 (ING-1) primer 224atgggctgga taaacaccta cactkgggag cctacatatg gtgatgactt caagggac 5822558DNAArtificial SequenceSynthesized H22-2NP3 (ING-1) primer 225atgggctgga taaacaccta cactscggag cctacatatg gtgatgactt caagggac 5822658DNAArtificial SequenceSynthesized H22-3B (ING-1) primer 226atgggctgga taaacaccta cactgaaarg cctacatatg gtgatgactt caagggac 5822758DNAArtificial SequenceSynthesized H22-3P1 (ING-1) primer 227atgggctgga taaacaccta cactgaawmc cctacatatg gtgatgactt caagggac 5822858DNAArtificial SequenceSynthesized H22-3P2 (ING-1) primer 228atgggctgga taaacaccta cactgaacas cctacatatg gtgatgactt caagggac 5822958DNAArtificial SequenceSynthesized H22-3A (ING-1) primer 229atgggctgga taaacaccta cactgaagas cctacatatg gtgatgactt caagggac 5823058DNAArtificial SequenceSynthesized H22-3NP1 (ING-1) primer 230atgggctgga taaacaccta cactgaantc cctacatatg gtgatgactt caagggac 5823158DNAArtificial SequenceSynthesized H22-3NP2 (ING-1) primer 231atgggctgga taaacaccta cactgaakgg cctacatatg gtgatgactt caagggac 5823258DNAArtificial SequenceSynthesized H22-3NP3 (ING-1) primer 232atgggctgga taaacaccta cactgaascg cctacatatg gtgatgactt caagggac 5823358DNAArtificial SequenceSynthesized H22-4B (ING-1) primer 233atgggctgga taaacaccta cactgaagag argacatatg gtgatgactt caagggac 5823458DNAArtificial SequenceSynthesized H22-4P1 (ING-1) primer 234atgggctgga taaacaccta cactgaagag wmcacatatg gtgatgactt caagggac 5823558DNAArtificial SequenceSynthesized H22-4P2 (ING-1) primer 235atgggctgga taaacaccta cactgaagag casacatatg gtgatgactt caagggac 5823658DNAArtificial SequenceSynthesized H22-4A (ING-1) primer 236atgggctgga taaacaccta cactgaagag gasacatatg gtgatgactt caagggac 5823758DNAArtificial SequenceSynthesized H22-4NP1 (ING-1) primer 237atgggctgga taaacaccta cactgaagag gasacatatg gtgatgactt caagggac 5823858DNAArtificial SequenceSynthesized H22-4NP2 (ING-1) primer 238atgggctgga taaacaccta cactgaagag kggacatatg gtgatgactt caagggac 5823958DNAArtificial SequenceSynthesized H22-4NP3 (ING-1) primer 239atgggctgga taaacaccta cactgaagag scgacatatg gtgatgactt caagggac 5824058DNAArtificial SequenceSynthesized H22-5B (ING-1) primer 240atgggctgga taaacaccta cactgaagag cctargtatg gtgatgactt caagggac 5824158DNAArtificial SequenceSynthesized H22-5P1 (ING-1) primer 241atgggctgga taaacaccta cactgaagag cctwmctatg gtgatgactt caagggac 5824258DNAArtificial SequenceSynthesized H22-5P2 (ING-1) primer 242atgggctgga taaacaccta cactgaagag cctcastatg gtgatgactt caagggac 5824358DNAArtificial SequenceSynthesized H22-5A (ING-1) primer 243atgggctgga taaacaccta cactgaagag cctgastatg gtgatgactt caagggac 5824458DNAArtificial SequenceSynthesized H22-5NP1 (ING-1) primer 244atgggctgga taaacaccta cactgaagag cctntctatg gtgatgactt caagggac 5824558DNAArtificial SequenceSynthesized H22-5NP2 (ING-1) primer 245atgggctgga taaacaccta cactgaagag cctkggtatg gtgatgactt caagggac 5824658DNAArtificial SequenceSynthesized H22-5NP3 (ING-1) primer 246atgggctgga taaacaccta cactgaagag cctscgtatg gtgatgactt caagggac 5824724DNAArtificial SequenceSynthesized H3-R (ING-1) primer 247aaatcttgca cagaaatatg tagc 2424853DNAArtificial SequenceSynthesized H3-1B (ING-1) primer 248gctacatatt tctgtgcaag atttargtct gctgtggact actggggtca agg 5324953DNAArtificial SequenceSynthesized H3-1P1 (ING-1) primer 249gctacatatt tctgtgcaag atttwmctct gctgtggact actggggtca agg 5325053DNAArtificial SequenceSynthesized H3-1P2 (ING-1) primer 250gctacatatt tctgtgcaag atttcastct gctgtggact actggggtca agg 5325153DNAArtificial SequenceSynthesized H3-1A (ING-1) primer 251gctacatatt tctgtgcaag atttgastct gctgtggact actggggtca agg 5325253DNAArtificial SequenceSynthesized H3-1NP1 (ING-1) primer

252gctacatatt tctgtgcaag atttntctct gctgtggact actggggtca agg 5325353DNAArtificial SequenceSynthesized H3-1NP2 (ING-1) primer 253gctacatatt tctgtgcaag atttkggtct gctgtggact actggggtca agg 5325453DNAArtificial SequenceSynthesized H3-1NP3 (ING-1) primer 254gctacatatt tctgtgcaag atttscgtct gctgtggact actggggtca agg 5325553DNAArtificial SequenceSynthesized H3-2B (ING-1) primer 255gctacatatt tctgtgcaag atttggcarg gctgtggact actggggtca agg 5325653DNAArtificial SequenceSynthesized H3-2P1 (ING-1) primer 256gctacatatt tctgtgcaag atttggcwmc gctgtggact actggggtca agg 5325753DNAArtificial SequenceSynthesized H3-2P2 (ING-1) primer 257gctacatatt tctgtgcaag atttggccas gctgtggact actggggtca agg 5325853DNAArtificial SequenceSynthesized H3-2A (ING-1) primer 258gctacatatt tctgtgcaag atttggcgas gctgtggact actggggtca agg 5325953DNAArtificial SequenceSynthesized H3-2NP1 (ING-1) primer 259gctacatatt tctgtgcaag atttggcntc gctgtggact actggggtca agg 5326053DNAArtificial SequenceSynthesized H3-2NP2 (ING-1) primer 260gctacatatt tctgtgcaag atttggckgg gctgtggact actggggtca agg 5326153DNAArtificial SequenceSynthesized H3-2NP3 (ING-1) primer 261gctacatatt tctgtgcaag atttggcscg gctgtggact actggggtca agg 5326253DNAArtificial SequenceSynthesized H3-3B (ING-1) primer 262gctacatatt tctgtgcaag atttggctct arggtggact actggggtca agg 5326353DNAArtificial SequenceSynthesized H3-3P1 (ING-1) primer 263gctacatatt tctgtgcaag atttggctct wmcgtggact actggggtca agg 5326453DNAArtificial SequenceSynthesized H3-3P2 (ING-1) primer 264gctacatatt tctgtgcaag atttggctct casgtggact actggggtca agg 5326553DNAArtificial SequenceSynthesized H3-3A (ING-1) primer 265gctacatatt tctgtgcaag atttggctct gasgtggact actggggtca agg 5326653DNAArtificial SequenceSynthesized H3-3NP1 (ING-1) primer 266gctacatatt tctgtgcaag atttggctct ntcgtggact actggggtca agg 5326753DNAArtificial SequenceSynthesized H3-3NP2 (ING-1) primer 267gctacatatt tctgtgcaag atttggctct kgggtggact actggggtca agg 5326853DNAArtificial SequenceSynthesized H3-3NP3 (ING-1) primer 268gctacatatt tctgtgcaag atttggctct scggtggact actggggtca agg 5326921DNAArtificial SequenceSynthesized H32-R (ING-1) primer 269cacagcagag ccaaatcttg c 2127046DNAArtificial SequenceSynthesized H32-1B (ING-1) primer 270gcaagatttg gctctgctgt gargtactgg ggtcaaggaa cctcgg 4627146DNAArtificial SequenceSynthesized H32-1P1 (ING-1) primer 271gcaagatttg gctctgctgt gwmctactgg ggtcaaggaa cctcgg 4627246DNAArtificial SequenceSynthesized H32-1P2 (ING-1) primer 272gcaagatttg gctctgctgt gcastactgg ggtcaaggaa cctcgg 4627346DNAArtificial SequenceSynthesized H32-1A (ING-1) primer 273gcaagatttg gctctgctgt ggastactgg ggtcaaggaa cctcgg 4627446DNAArtificial SequenceSynthesized H32-1NP1 (ING-1) primer 274gcaagatttg gctctgctgt gntctactgg ggtcaaggaa cctcgg 4627546DNAArtificial SequenceSynthesized H32-1NP2 (ING-1) primer 275gcaagatttg gctctgctgt gkggtactgg ggtcaaggaa cctcgg 4627646DNAArtificial SequenceSynthesized H32-1NP3 (ING-1) primer 276gcaagatttg gctctgctgt gscgtactgg ggtcaaggaa cctcgg 4627746DNAArtificial SequenceSynthesized H32-2B (ING-1) primer 277gcaagatttg gctctgctgt ggacargtgg ggtcaaggaa cctcgg 4627846DNAArtificial SequenceSynthesized H32-2P1 (ING-1) primer 278gcaagatttg gctctgctgt ggacwmctgg ggtcaaggaa cctcgg 4627946DNAArtificial SequenceSynthesized H32-2P2 (ING-1) primer 279gcaagatttg gctctgctgt ggaccastgg ggtcaaggaa cctcgg 4628046DNAArtificial SequenceSynthesized H32-2A (ING-1) primer 280gcaagatttg gctctgctgt ggacgastgg ggtcaaggaa cctcgg 4628146DNAArtificial SequenceSynthesized H32-2NP1 (ING-1) primer 281gcaagatttg gctctgctgt ggacntctgg ggtcaaggaa cctcgg 4628246DNAArtificial SequenceSynthesized H32-2NP2 (ING-1) primer 282gcaagatttg gctctgctgt ggackggtgg ggtcaaggaa cctcgg 4628346DNAArtificial SequenceSynthesized H32-2NP3 (ING-1) primer 283gcaagatttg gctctgctgt ggacscgtgg ggtcaaggaa cctcgg 4628422DNAArtificial SequenceSynthesized Ascl-F2 (ING-1) primer 284taataaggcg cgcctaacca tc 2228531DNAArtificial Sequencenotl-R (ING-1) primer 285agcggccgca caagatttgg gctcaactct c 3128620DNAArtificial SequenceSynthesized L1R (XPA-23) primer 286actcgcccga caaatgatgg 2028758DNAArtificial SequenceSynthesized L1-1B (XPA-23) primer 287ccatcatttg tcgggcgagt arggatatta acaggtggtt agcctggtat cagcagac 5828858DNAArtificial SequenceSynthesized L1-1P1 (XPA-23) primer 288ccatcatttg tcgggcgagt wmcgatatta acaggtggtt agcctggtat cagcagac 5828958DNAArtificial SequenceSynthesized L1-1P2 (XPA-23) primer 289ccatcatttg tcgggcgagt casgatatta acaggtggtt agcctggtat cagcagac 5829058DNAArtificial SequenceSynthesized L1-1A (XPA-23) primer 290ccatcatttg tcgggcgagt gasgatatta acaggtggtt agcctggtat cagcagac 5829158DNAArtificial SequenceSynthesized L1-1NP1 (XPA-23) primer 291ccatcatttg tcgggcgagt ntcgatatta acaggtggtt agcctggtat cagcagac 5829258DNAArtificial SequenceSynthesized L1-1NP2 (XPA-23) primer 292ccatcatttg tcgggcgagt kgggatatta acaggtggtt agcctggtat cagcagac 5829358DNAArtificial SequenceSynthesized L1-1NP3 (XPA-23) primer 293ccatcatttg tcgggcgagt scggatatta acaggtggtt agcctggtat cagcagac 5829458DNAArtificial SequenceSynthesized L1-2B (XPA-23) primer 294ccatcatttg tcgggcgagt cagargatta acaggtggtt agcctggtat cagcagac 5829558DNAArtificial SequenceSynthesized L1-2P1 (XPA-23) primer 295ccatcatttg tcgggcgagt cagwmcatta acaggtggtt agcctggtat cagcagac 5829658DNAArtificial SequenceSynthesized L1-2P2 (XPA-23) primer 296ccatcatttg tcgggcgagt cagcasatta acaggtggtt agcctggtat cagcagac 5829758DNAArtificial SequenceSynthesized L1-2A (XPA-23) primer 297ccatcatttg tcgggcgagt caggasatta acaggtggtt agcctggtat cagcagac 5829858DNAArtificial SequenceSynthesized L1-2NP1 (XPA-23) primer 298ccatcatttg tcgggcgagt cagntcatta acaggtggtt agcctggtat cagcagac 5829958DNAArtificial SequenceSynthesized L1-2NP2 (XPA-23) primer 299ccatcatttg tcgggcgagt cagkggatta acaggtggtt agcctggtat cagcagac 5830058DNAArtificial SequenceSynthesized L1-2NP3 (XPA-23) primer 300ccatcatttg tcgggcgagt cagscgatta acaggtggtt agcctggtat cagcagac 5830158DNAArtificial SequenceSynthesized L1-3B (XPA-23) primer 301ccatcatttg tcgggcgagt caggatatta rgaggtggtt agcctggtat cagcagac 5830258DNAArtificial SequenceSynthesized L1-3P1 (XPA-23) primer 302ccatcatttg tcgggcgagt caggatattw mcaggtggtt agcctggtat cagcagac 5830358DNAArtificial SequenceSynthesized L1-3P2 (XPA-23) primer 303ccatcatttg tcgggcgagt caggatattc asaggtggtt agcctggtat cagcagac 5830458DNAArtificial SequenceSynthesized L1-3A (XPA-23) primer 304ccatcatttg tcgggcgagt caggatattg asaggtggtt agcctggtat cagcagac 5830558DNAArtificial SequenceSynthesized L1-3NP1 (XPA-23) primer 305ccatcatttg tcgggcgagt caggatattn tcaggtggtt agcctggtat cagcagac 5830658DNAArtificial SequenceSynthesized L1-3NP2 (XPA-23) primer 306ccatcatttg tcgggcgagt caggatattk ggaggtggtt agcctggtat cagcagac 5830758DNAArtificial SequenceSynthesized L1-3NP3 (XPA-23) primer 307ccatcatttg tcgggcgagt caggatatts cgaggtggtt agcctggtat cagcagac 5830858DNAArtificial SequenceSynthesized L1-4B (XPA-23) primer 308ccatcatttg tcgggcgagt caggatatta acargtggtt agcctggtat cagcagac 5830958DNAArtificial SequenceSynthesized L1-4P1 (XPA-23) primer 309ccatcatttg tcgggcgagt caggatatta acwmctggtt agcctggtat cagcagac 5831058DNAArtificial SequenceSynthesized L1-4P2 (XPA-23) primer 310ccatcatttg tcgggcgagt caggatatta accastggtt agcctggtat cagcagac 5831158DNAArtificial SequenceSynthesized L1-4A (XPA-23) primer 311ccatcatttg tcgggcgagt caggatatta acgastggtt agcctggtat cagcagac 5831258DNAArtificial SequenceSynthesized L1-4NP1 (XPA-23) primer 312ccatcatttg tcgggcgagt caggatatta acntctggtt agcctggtat cagcagac 5831358DNAArtificial SequenceSynthesized L1-4NP2 (XPA-23) primer 313ccatcatttg tcgggcgagt caggatatta ackggtggtt agcctggtat cagcagac 5831458DNAArtificial SequenceSynthesized L1-4NP3 (XPA-23) primer 314ccatcatttg tcgggcgagt caggatatta acscgtggtt agcctggtat cagcagac 5831558DNAArtificial SequenceSynthesized L1-5B (XPA-23) primer 315ccatcatttg tcgggcgagt caggatatta acaggargtt agcctggtat cagcagac 5831658DNAArtificial SequenceSynthesized L1-5P1 (XPA-23) primer 316ccatcatttg tcgggcgagt caggatatta acaggwmctt agcctggtat cagcagac 5831758DNAArtificial SequenceSynthesized L1-5P2 (XPA-23) primer 317ccatcatttg tcgggcgagt caggatatta acaggcastt agcctggtat cagcagac 5831858DNAArtificial SequenceSynthesized L1-5NP1 (XPA-23) primer 318ccatcatttg tcgggcgagt caggatatta acaggntctt agcctggtat cagcagac 5831958DNAArtificial SequenceSynthesized L1-5NP2 (XPA-23) primer 319ccatcatttg tcgggcgagt caggatatta acaggkggtt agcctggtat cagcagac 5832058DNAArtificial SequenceSynthesized L1-5NP3 (XPA-23) primer 320ccatcatttg tcgggcgagt caggatatta acaggscgtt agcctggtat cagcagac 5832120DNAArtificial SequenceSynthesized L2R (XPA-23) primer 321gatcaggagc ttaggggcat 2032255DNAArtificial SequenceSynthesized L2-1B (XPA-23) primer 322atgcccctaa gctcctgatc argtctgcaa ccagtctgca aagtggggtc ccatc 5532355DNAArtificial SequenceSynthesized L2-1P1 (XPA-23) primer 323atgcccctaa gctcctgatc wmctctgcaa ccagtctgca aagtggggtc ccatc 5532455DNAArtificial SequenceSynthesized L2-1P2 (XPA-23) primer 324atgcccctaa gctcctgatc castctgcaa ccagtctgca aagtggggtc ccatc 5532555DNAArtificial SequenceSynthesized L2-1A (XPA-23) primer 325atgcccctaa gctcctgatc gastctgcaa ccagtctgca aagtggggtc ccatc 5532655DNAArtificial SequenceSynthesized L2-1NP1 (XPA-23) primer 326atgcccctaa gctcctgatc ntctctgcaa ccagtctgca aagtggggtc ccatc 5532755DNAArtificial SequenceSynthesized L2-1NP2 (XPA-23) primer 327atgcccctaa gctcctgatc kggtctgcaa ccagtctgca aagtggggtc ccatc 5532855DNAArtificial SequenceSynthesized L2-1NP3 (XPA-23) primer 328atgcccctaa gctcctgatc scgtctgcaa ccagtctgca aagtggggtc ccatc 5532955DNAArtificial SequenceSynthesized L2-2B (XPA-23) primer 329atgcccctaa gctcctgatc catarggcaa ccagtctgca aagtggggtc ccatc 5533055DNAArtificial SequenceSynthesized L2-2P1 (XPA-23) primer 330atgcccctaa gctcctgatc catwmcgcaa ccagtctgca aagtggggtc ccatc 5533155DNAArtificial SequenceSynthesized L2-2P2 (XPA-23) primer 331atgcccctaa gctcctgatc catcasgcaa ccagtctgca aagtggggtc ccatc 5533255DNAArtificial SequenceSynthesized L2-2A (XPA-23) primer 332atgcccctaa gctcctgatc catgasgcaa ccagtctgca aagtggggtc ccatc 5533355DNAArtificial SequenceSynthesized L2-2NP1 (XPA-23) primer 333atgcccctaa gctcctgatc catntcgcaa ccagtctgca aagtggggtc ccatc 5533455DNAArtificial SequenceSynthesized L2-2NP2 (XPA-23) primer 334atgcccctaa gctcctgatc catkgggcaa ccagtctgca aagtggggtc ccatc 5533555DNAArtificial SequenceSynthesized L2-2NP3 (XPA-23) primer 335atgcccctaa gctcctgatc catscggcaa ccagtctgca aagtggggtc ccatc 5533655DNAArtificial SequenceSynthesized L2-3B (XPA-23) primer 336atgcccctaa gctcctgatc cattctarga ccagtctgca aagtggggtc ccatc 5533755DNAArtificial SequenceSynthesized L2-3P1 (XPA-23) primer 337atgcccctaa gctcctgatc cattctwmca ccagtctgca aagtggggtc ccatc 5533855DNAArtificial SequenceSynthesized L2-3P2 (XPA-23) primer 338atgcccctaa gctcctgatc cattctcasa ccagtctgca aagtggggtc ccatc 5533955DNAArtificial SequenceSynthesized L2-3A (XPA-23) primer 339atgcccctaa gctcctgatc cattctgasa ccagtctgca aagtggggtc ccatc 5534055DNAArtificial SequenceSynthesized L2-3NP1 (XPA-23) primer 340atgcccctaa gctcctgatc cattctntca ccagtctgca aagtggggtc ccatc 5534155DNAArtificial SequenceSynthesized L2-3NP2 (XPA-23) primer 341atgcccctaa gctcctgatc cattctkgga ccagtctgca aagtggggtc ccatc 5534255DNAArtificial SequenceSynthesized L2-3NP3 (XPA-23) primer 342atgcccctaa gctcctgatc cattctscga ccagtctgca aagtggggtc ccatc 5534355DNAArtificial SequenceSynthesized L2-4B (XPA-23) primer 343atgcccctaa gctcctgatc cattctgcaa rgagtctgca aagtggggtc ccatc 5534455DNAArtificial SequenceSynthesized L2-4P1 (XPA-23) primer 344atgcccctaa gctcctgatc cattctgcaw mcagtctgca aagtggggtc ccatc 5534555DNAArtificial SequenceSynthesized L2-4P2 (XPA-23) primer 345atgcccctaa gctcctgatc cattctgcac asagtctgca aagtggggtc ccatc 5534655DNAArtificial SequenceSynthesized L2-4A (XPA-23) primer 346atgcccctaa gctcctgatc cattctgcag asagtctgca aagtggggtc ccatc 5534755DNAArtificial SequenceSynthesized L2-4NP1 (XPA-23) primer 347atgcccctaa gctcctgatc cattctgcan tcagtctgca aagtggggtc ccatc 5534855DNAArtificial SequenceSynthesized L2-4NP2 (XPA-23) primer 348atgcccctaa gctcctgatc cattctgcak ggagtctgca aagtggggtc ccatc 5534955DNAArtificial SequenceSynthesized L2-4NP3 (XPA-23) primer 349atgcccctaa gctcctgatc cattctgcas cgagtctgca aagtggggtc ccatc 5535055DNAArtificial SequenceSynthesized L2-5B (XPA-23) primer 350atgcccctaa gctcctgatc cattctgcaa ccargctgca aagtggggtc ccatc 5535155DNAArtificial SequenceSynthesized L2-5P1 (XPA-23) primer 351atgcccctaa gctcctgatc cattctgcaa ccwmcctgca aagtggggtc ccatc 5535255DNAArtificial SequenceSynthesized L2-5P2 (XPA-23) primer 352atgcccctaa gctcctgatc cattctgcaa cccasctgca aagtggggtc ccatc 5535355DNAArtificial SequenceSynthesized L2-5A (XPA-23) primer 353atgcccctaa gctcctgatc cattctgcaa ccgasctgca aagtggggtc ccatc 5535455DNAArtificial SequenceSynthesized L2-5NP1 (XPA-23) primer 354atgcccctaa gctcctgatc cattctgcaa ccntcctgca aagtggggtc ccatc 5535555DNAArtificial SequenceSynthesized L2-5NP2 (XPA-23) primer 355atgcccctaa gctcctgatc cattctgcaa cckggctgca aagtggggtc ccatc 5535655DNAArtificial SequenceSynthesized L2-5NP3 (XPA-23) primer 356atgcccctaa gctcctgatc cattctgcaa ccscgctgca aagtggggtc ccatc 5535721DNAArtificial SequenceSynthesized L3R (XPA-23) primer 357ctgctgacaa tagtaagttg c 2135857DNAArtificial SequenceSynthesized L3-1B (XPA-23) primer 358gcaacttact attgtcagca garggacagt ttcccgctca ctttcggcgg agggacc 5735957DNAArtificial SequenceSynthesized L3-1P1 (XPA-23) primer 359gcaacttact attgtcagca gwmcgacagt ttcccgctca ctttcggcgg agggacc 5736057DNAArtificial SequenceSynthesized L3-1P2 (XPA-23) primer 360gcaacttact attgtcagca gcasgacagt ttcccgctca ctttcggcgg agggacc 5736157DNAArtificial SequenceSynthesized L3-1A (XPA-23) primer 361gcaacttact attgtcagca ggasgacagt ttcccgctca ctttcggcgg agggacc 5736257DNAArtificial SequenceSynthesized L3-1NP1 (XPA-23) primer 362gcaacttact attgtcagca gntcgacagt ttcccgctca ctttcggcgg agggacc 5736357DNAArtificial SequenceSynthesized L3-1NP2 (XPA-23) primer 363gcaacttact attgtcagca gkgggacagt ttcccgctca ctttcggcgg agggacc 5736457DNAArtificial SequenceSynthesized L3-1NP3 (XPA-23) primer 364gcaacttact attgtcagca gscggacagt ttcccgctca ctttcggcgg agggacc 5736557DNAArtificial SequenceSynthesized L3-2B (XPA-23) primer 365gcaacttact attgtcagca ggctargagt ttcccgctca ctttcggcgg agggacc 5736657DNAArtificial SequenceSynthesized L3-2P1 (XPA-23) primer 366gcaacttact attgtcagca ggctwmcagt ttcccgctca ctttcggcgg agggacc 5736757DNAArtificial SequenceSynthesized L3-2P2 (XPA-23) primer 367gcaacttact attgtcagca ggctcasagt ttcccgctca ctttcggcgg agggacc 5736857DNAArtificial SequenceSynthesized L3-2A (XPA-23) primer 368gcaacttact attgtcagca ggctgasagt ttcccgctca ctttcggcgg agggacc 5736957DNAArtificial SequenceSynthesized L3-2NP1 (XPA-23) primer 369gcaacttact attgtcagca ggctntcagt ttcccgctca ctttcggcgg agggacc 5737057DNAArtificial SequenceSynthesized L3-2NP2 (XPA-23) primer 370gcaacttact attgtcagca ggctkggagt ttcccgctca ctttcggcgg agggacc 5737157DNAArtificial SequenceSynthesized L3-2NP3 (XPA-23) primer 371gcaacttact attgtcagca ggctscgagt ttcccgctca ctttcggcgg agggacc 5737257DNAArtificial SequenceSynthesized L3-3B (XPA-23) primer 372gcaacttact attgtcagca ggctgacarg ttcccgctca ctttcggcgg agggacc 5737357DNAArtificial SequenceSynthesized L3-3P1 (XPA-23) primer 373gcaacttact attgtcagca ggctgacwmc ttcccgctca ctttcggcgg agggacc 5737457DNAArtificial SequenceSynthesized L3-3P2 (XPA-23) primer 374gcaacttact attgtcagca ggctgaccas ttcccgctca ctttcggcgg agggacc 5737557DNAArtificial SequenceSynthesized L3-3A (XPA-23) primer 375gcaacttact attgtcagca ggctgacgas ttcccgctca ctttcggcgg agggacc 5737657DNAArtificial SequenceSynthesized L3-3NP1 (XPA-23) primer 376gcaacttact attgtcagca ggctgacntc ttcccgctca ctttcggcgg agggacc 5737757DNAArtificial SequenceSynthesized L3-3NP2 (XPA-23) primer 377gcaacttact attgtcagca ggctgackgg ttcccgctca ctttcggcgg agggacc

5737857DNAArtificial SequenceSynthesized L3-3NP3 (XPA-23) primer 378gcaacttact attgtcagca ggctgacscg ttcccgctca ctttcggcgg agggacc 5737957DNAArtificial SequenceSynthesized L3-4B (XPA-23) primer 379gcaacttact attgtcagca ggctgacagt argccgctca ctttcggcgg agggacc 5738057DNAArtificial SequenceSynthesized L3-4P1 (XPA-23) primer 380gcaacttact attgtcagca ggctgacagt wmcccgctca ctttcggcgg agggacc 5738157DNAArtificial SequenceSynthesized L3-4P2 (XPA-23) primer 381gcaacttact attgtcagca ggctgacagt casccgctca ctttcggcgg agggacc 5738257DNAArtificial SequenceSynthesized L3-4A (XPA-23) primer 382gcaacttact attgtcagca ggctgacagt gasccgctca ctttcggcgg agggacc 5738357DNAArtificial SequenceSynthesized L3-4NP1 (XPA-23) primer 383gcaacttact attgtcagca ggctgacagt ntcccgctca ctttcggcgg agggacc 5738457DNAArtificial SequenceSynthesized L3-4NP2 (XPA-23) primer 384gcaacttact attgtcagca ggctgacagt kggccgctca ctttcggcgg agggacc 5738557DNAArtificial SequenceSynthesized L3-4NP3 (XPA-23) primer 385gcaacttact attgtcagca ggctgacagt scgccgctca ctttcggcgg agggacc 5738657DNAArtificial SequenceSynthesized L3-5B (XPA-23) primer 386gcaacttact attgtcagca ggctgacagt ttcargctca ctttcggcgg agggacc 5738757DNAArtificial SequenceSynthesized L3-5P1 (XPA-23) primer 387gcaacttact attgtcagca ggctgacagt ttcwmcctca ctttcggcgg agggacc 5738857DNAArtificial SequenceSynthesized L3-5P2 (XPA-23) primer 388gcaacttact attgtcagca ggctgacagt ttccasctca ctttcggcgg agggacc 5738957DNAArtificial SequenceSynthesized L3-5A (XPA-23) primer 389gcaacttact attgtcagca ggctgacagt ttcgasctca ctttcggcgg agggacc 5739057DNAArtificial SequenceSynthesized L3-5NP1 (XPA-23) primer 390gcaacttact attgtcagca ggctgacagt ttcntcctca ctttcggcgg agggacc 5739157DNAArtificial SequenceSynthesized L3-5NP2 (XPA-23) primer 391gcaacttact attgtcagca ggctgacagt ttckggctca ctttcggcgg agggacc 5739257DNAArtificial SequenceSynthesized L3-5NP3 (XPA-23) primer 392gcaacttact attgtcagca ggctgacagt ttcscgctca ctttcggcgg agggacc 5739357DNAArtificial SequenceSynthesized L3-6B (XPA-23) primer 393gcaacttact attgtcagca ggctgacagt ttcccgarga ctttcggcgg agggacc 5739457DNAArtificial SequenceSynthesized L3-6P1 (XPA-23) primer 394gcaacttact attgtcagca ggctgacagt ttcccgwmca ctttcggcgg agggacc 5739557DNAArtificial SequenceSynthesized L3-6P2 (XPA-23) primer 395gcaacttact attgtcagca ggctgacagt ttcccgcasa ctttcggcgg agggacc 5739657DNAArtificial SequenceSynthesized L3-6A (XPA-23) primer 396gcaacttact attgtcagca ggctgacagt ttcccggasa ctttcggcgg agggacc 5739757DNAArtificial SequenceSynthesized L3-6NP1 (XPA-23) primer 397gcaacttact attgtcagca ggctgacagt ttcccgntca ctttcggcgg agggacc 5739857DNAArtificial SequenceSynthesized L3-6NP2 (XPA-23) primer 398gcaacttact attgtcagca ggctgacagt ttcccgkgga ctttcggcgg agggacc 5739957DNAArtificial SequenceSynthesized L3-6NP3 (XPA-23) primer 399gcaacttact attgtcagca ggctgacagt ttcccgscga ctttcggcgg agggacc 5740020DNAArtificial SequenceSynthesized H1R (XPA-23) primer 400gaatccggaa gcagcgcaag 2040154DNAArtificial SequenceSynthesized H1-1B (XPA-23) primer 401cttgcgctgc ttccggattc argttctcta agtactttat gttttgggtt cgcc 5440254DNAArtificial SequenceSynthesized H1-1P1 (XPA-23) primer 402cttgcgctgc ttccggattc wmcttctcta agtactttat gttttgggtt cgcc 5440354DNAArtificial SequenceSynthesized H1-1P2 (XPA-23) primer 403cttgcgctgc ttccggattc casttctcta agtactttat gttttgggtt cgcc 5440454DNAArtificial SequenceSynthesized H1-1A (XPA-23) primer 404cttgcgctgc ttccggattc gasttctcta agtactttat gttttgggtt cgcc 5440554DNAArtificial SequenceSynthesized H1-1NP1 (XPA-23) primer 405cttgcgctgc ttccggattc ntcttctcta agtactttat gttttgggtt cgcc 5440654DNAArtificial SequenceSynthesized H1-1NP3 (XPA-23) primer 406cttgcgctgc ttccggattc scgttctcta agtactttat gttttgggtt cgcc 5440754DNAArtificial SequenceSynthesized H1-2B (XPA-23) primer 407cttgcgctgc ttccggattc actttcarga agtactttat gttttgggtt cgcc 5440854DNAArtificial SequenceSynthesized H1-2P1 (XPA-23) primer 408cttgcgctgc ttccggattc actttcwmca agtactttat gttttgggtt cgcc 5440954DNAArtificial SequenceSynthesized H1-2P2 (XPA-23) primer 409cttgcgctgc ttccggattc actttccasa agtactttat gttttgggtt cgcc 5441054DNAArtificial SequenceSynthesized H1-2A (XPA-23) primer 410cttgcgctgc ttccggattc actttcgasa agtactttat gttttgggtt cgcc 5441154DNAArtificial SequenceSynthesized H1-2NP1 (XPA-23) primer 411cttgcgctgc ttccggattc actttcntca agtactttat gttttgggtt cgcc 5441254DNAArtificial SequenceSynthesized H1-2NP2 (XPA-23) primer 412cttgcgctgc ttccggattc actttckgga agtactttat gttttgggtt cgcc 5441354DNAArtificial SequenceSynthesized H1-2NP3 (XPA-23) primer 413cttgcgctgc ttccggattc actttcscga agtactttat gttttgggtt cgcc 5441454DNAArtificial SequenceSynthesized H1-3B (XPA-23) primer 414cttgcgctgc ttccggattc actttctcta rgtactttat gttttgggtt cgcc 5441554DNAArtificial SequenceSynthesized H1-3P1 (XPA-23) primer 415cttgcgctgc ttccggattc actttctctw mctactttat gttttgggtt cgcc 5441654DNAArtificial SequenceSynthesized H1-3P2 (XPA-23) primer 416cttgcgctgc ttccggattc actttctctc astactttat gttttgggtt cgcc 5441754DNAArtificial SequenceSynthesized H1-3A (XPA-23) primer 417cttgcgctgc ttccggattc actttctctg astactttat gttttgggtt cgcc 5441854DNAArtificial SequenceSynthesized H1-3NP1 (XPA-23) primer 418cttgcgctgc ttccggattc actttctctn tctactttat gttttgggtt cgcc 5441954DNAArtificial SequenceSynthesized H1-3NP2 (XPA-23) primer 419cttgcgctgc ttccggattc actttctctk ggtactttat gttttgggtt cgcc 5442054DNAArtificial SequenceSynthesized H1-3NP3 (XPA-23) primer 420cttgcgctgc ttccggattc actttctcts cgtactttat gttttgggtt cgcc 5442122DNAArtificial SequenceSynthesized H12R (XPA-23) primer 421cttagagaaa gtgaatccgg aa 2242253DNAArtificial SequenceSynthesized H12-1B (XPA-23) primer 422ttccggattc actttctcta agargtttat gttttgggtt cgccaagctc ctg 5342353DNAArtificial SequenceSynthesized H12-1P1 (XPA-23) primer 423ttccggattc actttctcta agwmctttat gttttgggtt cgccaagctc ctg 5342453DNAArtificial SequenceSynthesized H12-1P2 (XPA-23) primer 424ttccggattc actttctcta agcastttat gttttgggtt cgccaagctc ctg 5342553DNAArtificial SequenceSynthesized H12-1A (XPA-23) primer 425ttccggattc actttctcta aggastttat gttttgggtt cgccaagctc ctg 5342653DNAArtificial SequenceSynthesized H12-1NP1 (XPA-23) primer_ 426ttccggattc actttctcta agntctttat gttttgggtt cgccaagctc ctg 5342753DNAArtificial SequenceSynthesized H12-1NP2 (XPA-23) primer 427ttccggattc actttctcta agkggtttat gttttgggtt cgccaagctc ctg 5342853DNAArtificial SequenceSynthesized H12-1NP3 (XPA-23) primer 428ttccggattc actttctcta agscgtttat gttttgggtt cgccaagctc ctg 5342953DNAArtificial SequenceSynthesized H12-2B (XPA-23) primer 429ttccggattc actttctcta agtacargat gttttgggtt cgccaagctc ctg 5343053DNAArtificial SequenceSynthesized H12-2P1 (XPA-23) primer 430ttccggattc actttctcta agtacwmcat gttttgggtt cgccaagctc ctg 5343153DNAArtificial SequenceSynthesized H12-2P2 (XPA- 23) 431ttccggattc actttctcta agtaccasat gttttgggtt cgccaagctc ctg 5343253DNAArtificial SequenceSynthesized H12-2A (XPA-23) primer 432ttccggattc actttctcta agtacgasat gttttgggtt cgccaagctc ctg 5343353DNAArtificial SequenceSynthesized H12-2NP1 (XPA-23) primer 433ttccggattc actttctcta agtacntcat gttttgggtt cgccaagctc ctg 5343453DNAArtificial SequenceSynthesized H12-2NP2 (XPA-23) primer 434ttccggattc actttctcta agtackggat gttttgggtt cgccaagctc ctg 5343553DNAArtificial SequenceSynthesized H12-2NP3 (XPA-23) primer 435ttccggattc actttctcta agtacscgat gttttgggtt cgccaagctc ctg 5343653DNAArtificial SequenceSynthesized H12-3B (XPA-23) primer 436ttccggattc actttctcta agtactttat gargtgggtt cgccaagctc ctg 5343753DNAArtificial SequenceSynthesized H12-3P1 (XPA-23) primer 437ttccggattc actttctcta agtactttat gwmctgggtt cgccaagctc ctg 5343853DNAArtificial SequenceSynthesized H12-3P2 (XPA-23) primer 438ttccggattc actttctcta agtactttat gcastgggtt cgccaagctc ctg 5343953DNAArtificial SequenceSynthesized H12-3A (XPA-23) primer 439ttccggattc actttctcta agtactttat ggastgggtt cgccaagctc ctg 5344053DNAArtificial SequenceSynthesized H12-3NP1 (XPA-23) primer 440ttccggattc actttctcta agtactttat gntctgggtt cgccaagctc ctg 5344153DNAArtificial SequenceSynthesized H12-3NP2 (XPA-23) primer 441ttccggattc actttctcta agtactttat gkggtgggtt cgccaagctc ctg 5344253DNAArtificial SequenceSynthesized H12-3NP3 (XPA-23) primer 442ttccggattc actttctcta agtactttat gscgtgggtt cgccaagctc ctg 5344322DNAArtificial SequenceSynthesized H2R (XPA-23) primer 443agaaacccac tccaaacctt ta 2244457DNAArtificial SequenceSynthesized H2-1B (XPA-23) primer 444taaaggtttg gagtgggttt ctargatctc tccttctggt ggcatgactc gttatgc 5744557DNAArtificial SequenceSynthesized H2-1P1 (XPA-23) primer 445taaaggtttg gagtgggttt ctwmcatctc tccttctggt ggcatgactc gttatgc 5744657DNAArtificial SequenceSynthesized H2-1P2 (XPA-23) primer 446taaaggtttg gagtgggttt ctcasatctc tccttctggt ggcatgactc gttatgc 5744757DNAArtificial SequenceSynthesized H2-1A (XPA-23) primer 447taaaggtttg gagtgggttt ctgasatctc tccttctggt ggcatgactc gttatgc 5744857DNAArtificial SequenceSynthesized H2-1NP1 (XPA-23) primer 448taaaggtttg gagtgggttt ctntcatctc tccttctggt ggcatgactc gttatgc 5744957DNAArtificial SequenceSynthesized H2-1NP2 (XPA-23) primer 449taaaggtttg gagtgggttt ctkggatctc tccttctggt ggcatgactc gttatgc 5745057DNAArtificial SequenceSynthesized H2-1NP3 (XPA-23) primer 450taaaggtttg gagtgggttt ctscgatctc tccttctggt ggcatgactc gttatgc 5745157DNAArtificial SequenceSynthesized H2-2B (XPA-23) primer 451taaaggtttg gagtgggttt ctgttargtc tccttctggt ggcatgactc gttatgc 5745257DNAArtificial SequenceSynthesized H2-2P1 (XPA-23) primer 452taaaggtttg gagtgggttt ctgttwmctc tccttctggt ggcatgactc gttatgc 5745357DNAArtificial SequenceSynthesized H2-2P2 (XPA-23) primer 453taaaggtttg gagtgggttt ctgttcastc tccttctggt ggcatgactc gttatgc 5745457DNAArtificial SequenceSynthesized H2-2A (XPA-23) primer 454taaaggtttg gagtgggttt ctgttgastc tccttctggt ggcatgactc gttatgc 5745557DNAArtificial SequenceSynthesized H2-2NP1 (XPA-23) primer 455taaaggtttg gagtgggttt ctgttntctc tccttctggt ggcatgactc gttatgc 5745657DNAArtificial SequenceSynthesized H2-2NP2 (XPA-23) primer 456taaaggtttg gagtgggttt ctgttkggtc tccttctggt ggcatgactc gttatgc 5745757DNAArtificial SequenceSynthesized H2-2NP3 (XPA-23) primer 457taaaggtttg gagtgggttt ctgttscgtc tccttctggt ggcatgactc gttatgc 5745857DNAArtificial SequenceSynthesized H2-3B (XPA-23) primer 458taaaggtttg gagtgggttt ctgttatcar gccttctggt ggcatgactc gttatgc 5745957DNAArtificial SequenceSynthesized H2-3P1 (XPA-23) primer 459taaaggtttg gagtgggttt ctgttatcwm cccttctggt ggcatgactc gttatgc 5746057DNAArtificial SequenceSynthesized H2-3P2 (XPA-23) primer 460taaaggtttg gagtgggttt ctgttatcca sccttctggt ggcatgactc gttatgc 5746157DNAArtificial SequenceSynthesized H2-3A (XPA-23) primer 461taaaggtttg gagtgggttt ctgttatcga sccttctggt ggcatgactc gttatgc 5746257DNAArtificial SequenceSynthesized H2-3NP1 (XPA-23) primer 462taaaggtttg gagtgggttt ctgttatcnt cccttctggt ggcatgactc gttatgc 5746357DNAArtificial SequenceSynthesized H2-3NP2 (XPA-23) primer 463taaaggtttg gagtgggttt ctgttatckg gccttctggt ggcatgactc gttatgc 5746457DNAArtificial SequenceSynthesized H2-3NP3 (XPA-23) primer 464taaaggtttg gagtgggttt ctgttatcsc gccttctggt ggcatgactc gttatgc 5746557DNAArtificial SequenceSynthesized H2-4B (XPA-23) primer 465taaaggtttg gagtgggttt ctgttatctc targtctggt ggcatgactc gttatgc 5746657DNAArtificial SequenceSynthesized H2-4P1 (XPA-23) primer 466taaaggtttg gagtgggttt ctgttatctc twmctctggt ggcatgactc gttatgc 5746757DNAArtificial SequenceSynthesized H2-4P2 (XPA-23) primer 467taaaggtttg gagtgggttt ctgttatctc tcastctggt ggcatgactc gttatgc 5746857DNAArtificial SequenceSynthesized H2-4A (XPA-23) primer 468taaaggtttg gagtgggttt ctgttatctc tgastctggt ggcatgactc gttatgc 5746957DNAArtificial SequenceSynthesized H2-4NP1 (XPA-23) primer 469taaaggtttg gagtgggttt ctgttatctc tntctctggt ggcatgactc gttatgc 5747057DNAArtificial SequenceSynthesized H2-4NP2 (XPA-23) primer 470taaaggtttg gagtgggttt ctgttatctc tkggtctggt ggcatgactc gttatgc 5747157DNAArtificial SequenceSynthesized H2-4NP3 (XPA-23) primer 471taaaggtttg gagtgggttt ctgttatctc tscgtctggt ggcatgactc gttatgc 5747257DNAArtificial SequenceSynthesized H2-5B (XPA-23) primer 472taaaggtttg gagtgggttt ctgttatctc tcctargggt ggcatgactc gttatgc 5747357DNAArtificial SequenceSynthesized H2-5P1 (XPA-23) primer 473taaaggtttg gagtgggttt ctgttatctc tcctwmcggt ggcatgactc gttatgc 5747457DNAArtificial SequenceSynthesized H2-5P2 (XPA-23) primer 474taaaggtttg gagtgggttt ctgttatctc tcctcasggt ggcatgactc gttatgc 5747557DNAArtificial SequenceSynthesized H2-5A (XPA-23) primer 475taaaggtttg gagtgggttt ctgttatctc tcctgasggt ggcatgactc gttatgc 5747657DNAArtificial SequenceSynthesized H2-5NP1 (XPA-23) primer 476taaaggtttg gagtgggttt ctgttatctc tcctntcggt ggcatgactc gttatgc 5747757DNAArtificial SequenceSynthesized H2-5NP2 (XPA-23) primer 477taaaggtttg gagtgggttt ctgttatctc tcctkggggt ggcatgactc gttatgc 5747857DNAArtificial SequenceSynthesized H2-5NP3 (XPA-23) primer 478taaaggtttg gagtgggttt ctgttatctc tcctscgggt ggcatgactc gttatgc 5747922DNAArtificial SequenceSynthesized H22-R (XPA-23) primer 479agaaggagag ataacagaaa cc 2248059DNAArtificial SequenceSynthesized H22-1P1 (XPA-23) primer 480ggtttctgtt atctctcctt ctwmcggcat gactcgttat gctgactccg ttaaaggtc 5948159DNAArtificial SequenceSynthesized H22-1P2 (XPA-23) primer 481ggtttctgtt atctctcctt ctcasggcat gactcgttat gctgactccg ttaaaggtc 5948259DNAArtificial SequenceSynthesized H22-1A (XPA-23) primer 482ggtttctgtt atctctcctt ctgasggcat gactcgttat gctgactccg ttaaaggtc 5948359DNAArtificial SequenceSynthesized H22-1NP1 (XPA-23) primer 483ggtttctgtt atctctcctt ctntcggcat gactcgttat gctgactccg ttaaaggtc 5948459DNAArtificial SequenceSynthesized H22-1NP2 (XPA-23) primer 484ggtttctgtt atctctcctt ctkggggcat gactcgttat gctgactccg ttaaaggtc 5948559DNAArtificial SequenceSynthesized H22-1NP3 (XPA-23) primer 485ggtttctgtt atctctcctt ctscgggcat gactcgttat gctgactccg ttaaaggtc 5948659DNAArtificial SequenceSynthesized H22-2B (XPA-23) primer 486ggtttctgtt atctctcctt ctggtargat gactcgttat gctgactccg ttaaaggtc 5948759DNAArtificial SequenceSynthesized H22-2P1 (XPA-23) primer 487ggtttctgtt atctctcctt ctggtwmcat gactcgttat gctgactccg ttaaaggtc 5948859DNAArtificial SequenceSynthesized H22-2P2 (XPA-23) primer 488ggtttctgtt atctctcctt ctggtcasat gactcgttat gctgactccg ttaaaggtc 5948959DNAArtificial SequenceSynthesized H22-2A (XPA-23) primer 489ggtttctgtt atctctcctt ctggtgasat gactcgttat gctgactccg ttaaaggtc 5949059DNAArtificial SequenceSynthesized H22-2NP1 (XPA-23) primer 490ggtttctgtt atctctcctt ctggtntcat gactcgttat gctgactccg ttaaaggtc 5949159DNAArtificial SequenceSynthesized H22-2NP2 (XPA-23) primer 491ggtttctgtt atctctcctt ctggtkggat gactcgttat gctgactccg ttaaaggtc 5949259DNAArtificial SequenceSynthesized H22-2NP3 (XPA-23) primer 492ggtttctgtt atctctcctt ctggtscgat gactcgttat gctgactccg ttaaaggtc 5949359DNAArtificial SequenceSynthesized H22-3B (XPA-23) primer 493ggtttctgtt atctctcctt ctggtggcar gactcgttat gctgactccg ttaaaggtc 5949459DNAArtificial SequenceSynthesized H22-3P1 (XPA-23) primer 494ggtttctgtt atctctcctt ctggtggcwm cactcgttat gctgactccg ttaaaggtc 5949559DNAArtificial SequenceSynthesized H22-3P2 (XPA-23) primer 495ggtttctgtt atctctcctt ctggtggcca sactcgttat gctgactccg ttaaaggtc 5949659DNAArtificial SequenceSynthesized H22-3A (XPA-23) primer 496ggtttctgtt atctctcctt ctggtggcga sactcgttat gctgactccg ttaaaggtc 5949759DNAArtificial SequenceSynthesized H22-3NP1 (XPA-23) primer 497ggtttctgtt atctctcctt ctggtggcnt cactcgttat gctgactccg ttaaaggtc 5949859DNAArtificial SequenceSynthesized H22-3NP2 (XPA-23) primer 498ggtttctgtt atctctcctt ctggtggckg gactcgttat gctgactccg ttaaaggtc 5949959DNAArtificial SequenceSynthesized H22-3NP3 (XPA-23) primer 499ggtttctgtt atctctcctt ctggtggcsc gactcgttat gctgactccg ttaaaggtc 5950059DNAArtificial SequenceSynthesized H22-4B (XPA-23) primer 500ggtttctgtt atctctcctt ctggtggcat gargcgttat gctgactccg ttaaaggtc 5950159DNAArtificial SequenceSynthesized H22-4P1 (XPA-23) primer 501ggtttctgtt atctctcctt ctggtggcat gwmccgttat gctgactccg ttaaaggtc 5950259DNAArtificial SequenceSynthesized H22-4P2 (XPA-23) primer 502ggtttctgtt atctctcctt ctggtggcat gcascgttat gctgactccg ttaaaggtc 5950359DNAArtificial SequenceSynthesized H22-4A (XPA-23) primer

503ggtttctgtt atctctcctt ctggtggcat ggascgttat gctgactccg ttaaaggtc 5950459DNAArtificial SequenceSynthesized H22-4NP1 (XPA-23) primer 504ggtttctgtt atctctcctt ctggtggcat gntccgttat gctgactccg ttaaaggtc 5950559DNAArtificial SequenceSynthesized H22-4NP2 (XPA-23) primer 505ggtttctgtt atctctcctt ctggtggcat gkggcgttat gctgactccg ttaaaggtc 5950659DNAArtificial SequenceSynthesized H22-4NP3 (XPA-23) primer 506ggtttctgtt atctctcctt ctggtggcat gscgcgttat gctgactccg ttaaaggtc 5950759DNAArtificial SequenceSynthesized H22-5B (XPA-23) primer 507ggtttctgtt atctctcctt ctggtggcat gactargtat gctgactccg ttaaaggtc 5950859DNAArtificial SequenceSynthesized H22-5P1 (XPA-23) primer 508ggtttctgtt atctctcctt ctggtggcat gactwmctat gctgactccg ttaaaggtc 5950959DNAArtificial SequenceSynthesized H22-5P2 (XPA-23) primer 509ggtttctgtt atctctcctt ctggtggcat gactcastat gctgactccg ttaaaggtc 5951059DNAArtificial SequenceSynthesized H22-5A (XPA-23) primer 510ggtttctgtt atctctcctt ctggtggcat gactgastat gctgactccg ttaaaggtc 5951159DNAArtificial SequenceSynthesized H22-5NP1 (XPA-23) primer 511ggtttctgtt atctctcctt ctggtggcat gactntctat gctgactccg ttaaaggtc 5951259DNAArtificial SequenceSynthesized H22-5NP2 (XPA-23) primer 512ggtttctgtt atctctcctt ctggtggcat gactkggtat gctgactccg ttaaaggtc 5951359DNAArtificial SequenceSynthesized H22-5NP3 (XPA-23) primer 513ggtttctgtt atctctcctt ctggtggcat gactscgtat gctgactccg ttaaaggtc 5951421DNAArtificial SequenceSynthesized H3-R (XPA-23) primer 514tctcgcacaa tagtagactg c 2151556DNAArtificial SequenceSynthesized H3-1B (XPA-23) primer 515gcagtctact attgtgcgag aargggctac ggtggtaact ctgactactg gggcca 5651656DNAArtificial SequenceSynthesized H3-1P1 (XPA-23) primer 516gcagtctact attgtgcgag awmcggctac ggtggtaact ctgactactg gggcca 5651756DNAArtificial SequenceSynthesized H3-1P2 (XPA-23) primer 517gcagtctact attgtgcgag acasggctac ggtggtaact ctgactactg gggcca 5651856DNAArtificial SequenceSynthesized H3-1A (XPA-23) primer 518gcagtctact attgtgcgag agasggctac ggtggtaact ctgactactg gggcca 5651956DNAArtificial SequenceSynthesized H3-1NP1 (XPA-23) primer 519gcagtctact attgtgcgag antcggctac ggtggtaact ctgactactg gggcca 5652056DNAArtificial SequenceSynthesized H3-1NP2 (XPA-23) primer 520gcagtctact attgtgcgag akggggctac ggtggtaact ctgactactg gggcca 5652156DNAArtificial SequenceSynthesized H3-1NP3 (XPA-23) primer 521gcagtctact attgtgcgag ascgggctac ggtggtaact ctgactactg gggcca 5652256DNAArtificial SequenceSynthesized H3-2B (XPA-23) primer 522gcagtctact attgtgcgag agtcargtac ggtggtaact ctgactactg gggcca 5652356DNAArtificial SequenceSynthesized H3-2P1 (XPA-23) primer 523gcagtctact attgtgcgag agtcwmctac ggtggtaact ctgactactg gggcca 5652456DNAArtificial SequenceSynthesized H3-2P2 (XPA-23) primer 524gcagtctact attgtgcgag agtccastac ggtggtaact ctgactactg gggcca 5652556DNAArtificial SequenceSynthesized H3-2A (XPA-23) primer 525gcagtctact attgtgcgag agtcgastac ggtggtaact ctgactactg gggcca 5652656DNAArtificial SequenceSynthesized H3-2NP1 (XPA-23) primer 526gcagtctact attgtgcgag agtcntctac ggtggtaact ctgactactg gggcca 5652756DNAArtificial SequenceSynthesized H3-2NP2 (XPA-23) primer 527gcagtctact attgtgcgag agtckggtac ggtggtaact ctgactactg gggcca 5652856DNAArtificial SequenceSynthesized H3-2NP3 (XPA-23) primer 528gcagtctact attgtgcgag agtcscgtac ggtggtaact ctgactactg gggcca 5652956DNAArtificial SequenceSynthesized H3-3B (XPA-23) primer 529gcagtctact attgtgcgag agtcggcarg ggtggtaact ctgactactg gggcca 5653056DNAArtificial SequenceSynthesized H3-3P1 (XPA-23) primer 530gcagtctact attgtgcgag agtcggcwmc ggtggtaact ctgactactg gggcca 5653156DNAArtificial SequenceSynthesized H3-3P2 (XPA-23) primer 531gcagtctact attgtgcgag agtcggccas ggtggtaact ctgactactg gggcca 5653256DNAArtificial SequenceSynthesized H3-3A (XPA-23) primer 532gcagtctact attgtgcgag agtcggcgas ggtggtaact ctgactactg gggcca 5653356DNAArtificial SequenceSynthesized H3-3NP1 (XPA-23) primer 533gcagtctact attgtgcgag agtcggcntc ggtggtaact ctgactactg gggcca 5653456DNAArtificial SequenceSynthesized H3-3NP2 (XPA-23) primer 534gcagtctact attgtgcgag agtcggckgg ggtggtaact ctgactactg gggcca 5653556DNAArtificial SequenceSynthesized H3-3NP3 (XPA-23) primer 535gcagtctact attgtgcgag agtcggcscg ggtggtaact ctgactactg gggcca 5653656DNAArtificial SequenceSynthesized H3-4B (XPA-23) primer 536gcagtctact attgtgcgag agtcggctac argggtaact ctgactactg gggcca 5653756DNAArtificial SequenceSynthesized H3-4P1 (XPA-23) primer 537gcagtctact attgtgcgag agtcggctac wmcggtaact ctgactactg gggcca 5653856DNAArtificial SequenceSynthesized H3-4P2 (XPA-23) primer 538gcagtctact attgtgcgag agtcggctac casggtaact ctgactactg gggcca 5653956DNAArtificial SequenceSynthesized H3-4A (XPA-23) primer 539gcagtctact attgtgcgag agtcggctac gasggtaact ctgactactg gggcca 5654056DNAArtificial SequenceSynthesized H3-4NP1 (XPA-23) primer 540gcagtctact attgtgcgag agtcggctac ntcggtaact ctgactactg gggcca 5654156DNAArtificial SequenceSynthesized H3-4NP2 (XPA-23) primer 541gcagtctact attgtgcgag agtcggctac kggggtaact ctgactactg gggcca 5654256DNAArtificial SequenceSynthesized H3-4NP3 (XPA-23) primer 542gcagtctact attgtgcgag agtcggctac scgggtaact ctgactactg gggcca 5654356DNAArtificial SequenceSynthesized H3-5B (XPA-23) primer 543gcagtctact attgtgcgag agtcggctac ggtargaact ctgactactg gggcca 5654456DNAArtificial SequenceSynthesized H3-5P1 (XPA-23) primer 544gcagtctact attgtgcgag agtcggctac ggtwmcaact ctgactactg gggcca 5654556DNAArtificial SequenceSynthesized H3-5P2 (XPA-23) primer 545gcagtctact attgtgcgag agtcggctac ggtcasaact ctgactactg gggcca 5654656DNAArtificial SequenceSynthesized H3-5A (XPA-23) primer 546gcagtctact attgtgcgag agtcggctac ggtgasaact ctgactactg gggcca 5654756DNAArtificial SequenceSynthesized H3-5NP1 (XPA-23) primer 547gcagtctact attgtgcgag agtcggctac ggtntcaact ctgactactg gggcca 5654856DNAArtificial SequenceSynthesized H3-5NP2 (XPA-23) primer 548gcagtctact attgtgcgag agtcggctac ggtkggaact ctgactactg gggcca 5654956DNAArtificial SequenceSynthesized H3-5NP3 (XPA-23) primer 549gcagtctact attgtgcgag agtcggctac ggtscgaact ctgactactg gggcca 5655019DNAArtificial SequenceSynthesized H32R (XPA-23) primer 550accaccgtag ccgactctc 1955149DNAArtificial SequenceSynthesized H32-1B (XPA-23) primer 551gagagtcggc tacggtggta rgtctgacta ctggggccag ggaaccctg 4955249DNAArtificial SequenceSynthesized H32-1P1 (XPA-23) primer 552gagagtcggc tacggtggtw mctctgacta ctggggccag ggaaccctg 4955349DNAArtificial SequenceSynthesized H32-1P2 (XPA-23) primer 553gagagtcggc tacggtggtc astctgacta ctggggccag ggaaccctg 4955449DNAArtificial SequenceSynthesized H32-1A (XPA-23) primer 554gagagtcggc tacggtggtg astctgacta ctggggccag ggaaccctg 4955549DNAArtificial SequenceSynthesized H32-1NP1 (XPA-23) primer 555gagagtcggc tacggtggtn tctctgacta ctggggccag ggaaccctg 4955649DNAArtificial SequenceSynthesized H32-1NP2 (XPA-23) primer 556gagagtcggc tacggtggtk ggtctgacta ctggggccag ggaaccctg 4955749DNAArtificial SequenceSynthesized H32-1NP3 (XPA-23) primer 557gagagtcggc tacggtggts cgtctgacta ctggggccag ggaaccctg 4955849DNAArtificial SequenceSynthesized H32-2B (XPA-23) primer 558gagagtcggc tacggtggta acarggacta ctggggccag ggaaccctg 4955949DNAArtificial SequenceSynthesized H32-2P1 (XPA-23) primer 559gagagtcggc tacggtggta acwmcgacta ctggggccag ggaaccctg 4956049DNAArtificial SequenceSynthesized H32-2P2 (XPA-23) primer 560gagagtcggc tacggtggta accasgacta ctggggccag ggaaccctg 4956149DNAArtificial SequenceSynthesized H32-2A (XPA-23) primer 561gagagtcggc tacggtggta acgasgacta ctggggccag ggaaccctg 4956249DNAArtificial SequenceSynthesized H32-2NP1 (XPA-23) primer 562gagagtcggc tacggtggta acntcgacta ctggggccag ggaaccctg 4956349DNAArtificial SequenceSynthesized H32-2NP2 (XPA-23) primer 563gagagtcggc tacggtggta ackgggacta ctggggccag ggaaccctg 4956449DNAArtificial SequenceSynthesized H32-2NP3 (XPA-23) primer 564gagagtcggc tacggtggta acscggacta ctggggccag ggaaccctg 4956549DNAArtificial SequenceSynthesized H32-3B (XPA-23) primer 565gagagtcggc tacggtggta actctargta ctggggccag ggaaccctg 4956649DNAArtificial SequenceSynthesized H32-3P1 (XPA-23) primer 566gagagtcggc tacggtggta actctwmcta ctggggccag ggaaccctg 4956749DNAArtificial SequenceSynthesized H32-3P2 (XPA-23) primer 567gagagtcggc tacggtggta actctcasta ctggggccag ggaaccctg 4956849DNAArtificial SequenceSynthesized H32-3A (XPA-23) primer 568gagagtcggc tacggtggta actctgasta ctggggccag ggaaccctg 4956949DNAArtificial SequenceSynthesized H32-3NP1 (XPA-23) primer 569gagagtcggc tacggtggta actctntcta ctggggccag ggaaccctg 4957049DNAArtificial SequenceSynthesized H32-3NP2 (XPA-23) primer 570gagagtcggc tacggtggta actctkggta ctggggccag ggaaccctg 4957149DNAArtificial SequenceSynthesized H32-3NP3 (XPA-23) primer 571gagagtcggc tacggtggta actctscgta ctggggccag ggaaccctg 4957249DNAArtificial SequenceSynthesized H32-4B (XPA-23) primer 572gagagtcggc tacggtggta actctgacar gtggggccag ggaaccctg 4957349DNAArtificial SequenceSynthesized H32-4P1 (XPA-23) primer 573gagagtcggc tacggtggta actctgacwm ctggggccag ggaaccctg 4957449DNAArtificial SequenceSynthesized H32-4P2 (XPA-23) primer 574gagagtcggc tacggtggta actctgacca stggggccag ggaaccctg 4957549DNAArtificial SequenceSynthesized H32-4A (XPA-23) primer 575gagagtcggc tacggtggta actctgacga stggggccag ggaaccctg 4957649DNAArtificial SequenceSynthesized H32-4NP1 (XPA-23) primer 576gagagtcggc tacggtggta actctgacnt ctggggccag ggaaccctg 4957749DNAArtificial SequenceSynthesized H32-4NP2 (XPA-23) primer 577gagagtcggc tacggtggta actctgackg gtggggccag ggaaccctg 4957849DNAArtificial SequenceSynthesized H32-4NP3 (XPA-23) primer 578gagagtcggc tacggtggta actctgacsc gtggggccag ggaaccctg 49579675DNAArtificial SequenceSynthesized ING-1 (heavy chain) 579cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg cttctggata taccttcaca aaatatggaa tgaactgggt gaagcaggct 120ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactgaaga gccaacatat 180ggtgatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgccaat 240ttgcagatca acaacctcaa aagtgaggac acggctacat atttctgtgc aagatttggc 300tctgctgtgg actactgggg tcaaggaacc tcggtcaccg tctcctcagc cagcacaaag 360ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 420ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 480gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 540ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 600gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 660aaaactcaca catga 675580675DNAArtificial SequenceSynthesized ING-1 (light chain) 580caaccagcga tggcggatat tgtgatgacg caggctgcat tctccaatcc agtcactctt 60ggaacatcag gttccatctc ctgcaggtct agtaagagtc tcctacatag taatggcatc 120acttatttgt attggtatct gcagaagcca ggccagtctc ctcagctcct gatttatcag 180atgtccaacc ttgcctcagg agtcccagac aggttcagta gcagtgggtc aggaactgat 240ttcacactga gaatcagcag agtggaggct gaggatgtgg gtgtttatta ctgtgctcaa 300aatctagaac ttcctcggac gttcggtgga ggcaccaagc ttgagatgaa acgaactgtg 360gctgcaccat ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc 420tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg 480gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac 540agcacctaca gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa 600gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac 660aggggagagt gttag 675581354DNAArtificial SequenceSynthesized IL-1 (XPA-23, heavy chain) 581gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct aagtacttta tgttttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctgtt atctctcctt ctggtggcat gactcgttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagtcggc 300tacggtggta actctgacta ctggggccag ggaaccctgg tcaccgtctc aagc 354582324DNAArtificial SequenceSynthesized IL-1 (XPA-23, kappa chain) 582caagacatcc agatgaccca gtctccctct tccgtgtctg catctgtagg agacagactc 60accatcattt gtcgggcgag tcaggatatt aacaggtggt tagcctggta tcagcagaca 120ccagggaatg cccctaagct cctgatccat tctgcaacca gtctgcaaag tggggtccca 180tcaaggttta gcggcagtgg atctgggaca gatttcactc tcaccatcaa cagcctgcag 240cctgaagatt ttgcaactta ctattgtcag caggctgaca gtttcccgct cactttcggc 300ggagggacca aggtggagat caaa 32458359DNAArtificial SequenceSynthesized L3-1NHT (ING1) 583ggtgtttatt actgtgctca aaatnhtgaa cttcctcgga cgttcggtgg aggcaccaa 5958459DNAArtificial SequenceSynthesized L3-1VAA (ING1) 584ggtgtttatt actgtgctca aaatvaagaa cttcctcgga cgttcggtgg aggcaccaa 5958559DNAArtificial SequenceSynthesized L3-1BGG (ING1) 585ggtgtttatt actgtgctca aaatbgggaa cttcctcgga cgttcggtgg aggcaccaa 5958659DNAArtificial SequenceSynthesized L3-2NHT (ING1) 586ggtgtttatt actgtgctca aaatctanht cttcctcgga cgttcggtgg aggcaccaa 5958759DNAArtificial SequenceSynthesized L3-2VAA (ING1) 587ggtgtttatt actgtgctca aaatctavaa cttcctcgga cgttcggtgg aggcaccaa 5958859DNAArtificial SequenceSynthesized L3-2BGG (ING1) 588ggtgtttatt actgtgctca aaatctabgg cttcctcgga cgttcggtgg aggcaccaa 5958959DNAArtificial SequenceSynthesized L3-3NHT (ING1) 589ggtgtttatt actgtgctca aaatctagaa nhtcctcgga cgttcggtgg aggcaccaa 5959059DNAArtificial SequenceSynthesized L3-3VAA (ING1) 590ggtgtttatt actgtgctca aaatctagaa vaacctcgga cgttcggtgg aggcaccaa 5959159DNAArtificial SequenceSynthesized L3-3BGG (ING1) 591ggtgtttatt actgtgctca aaatctagaa bggcctcgga cgttcggtgg aggcaccaa 5959259DNAArtificial SequenceSynthesized L3-4NHT (ING1) 592ggtgtttatt actgtgctca aaatctagaa cttnhtcgga cgttcggtgg aggcaccaa 5959359DNAArtificial SequenceSynthesized L3-4VAA (ING1) 593ggtgtttatt actgtgctca aaatctagaa cttvaacgga cgttcggtgg aggcaccaa 5959459DNAArtificial SequenceSynthesized L3-4BGG (ING1) 594ggtgtttatt actgtgctca aaatctagaa cttbggcgga cgttcggtgg aggcaccaa 5959559DNAArtificial SequenceSynthesized L3-5NHT (ING1) 595ggtgtttatt actgtgctca aaatctagaa cttcctnhta cgttcggtgg aggcaccaa 5959659DNAArtificial SequenceSynthesized L3-5VAA (ING1) 596ggtgtttatt actgtgctca aaatctagaa cttcctvaaa cgttcggtgg aggcaccaa 5959759DNAArtificial SequenceSynthesized L3-5BGG (ING1) 597ggtgtttatt actgtgctca aaatctagaa cttcctbgga cgttcggtgg aggcaccaa 5959855DNAArtificial SequenceSynthesized L1-1NHT (ING1) 598ccatctcctg ccgctctagt nhtagtctcc tacatagtaa tggcatcact tattt 5559955DNAArtificial SequenceSynthesized L1-1VAA (ING1) 599ccatctcctg ccgctctagt vaaagtctcc tacatagtaa tggcatcact tattt 5560055DNAArtificial SequenceSynthesized L1-1BGG (ING1) 600ccatctcctg ccgctctagt bggagtctcc tacatagtaa tggcatcact tattt 5560155DNAArtificial SequenceSynthesized L1-2NHT (ING1) 601ccatctcctg ccgctctagt aagnhtctcc tacatagtaa tggcatcact tattt 5560255DNAArtificial SequenceSynthesized L1-2VAA (ING1) 602ccatctcctg ccgctctagt aagvaactcc tacatagtaa tggcatcact tattt 5560355DNAArtificial SequenceSynthesized L1-2BGG (ING1) 603ccatctcctg ccgctctagt aagbggctcc tacatagtaa tggcatcact tattt 5560455DNAArtificial SequenceSynthesized L1-3NHT (ING1) 604ccatctcctg ccgctctagt aagagtnhtc tacatagtaa tggcatcact tattt 5560555DNAArtificial SequenceSynthesized L1-3VAA (ING1) 605ccatctcctg ccgctctagt aagagtvaac tacatagtaa tggcatcact tattt 5560655DNAArtificial SequenceSynthesized L1-3BGG (ING1) 606ccatctcctg ccgctctagt aagagtbggc tacatagtaa tggcatcact tattt 5560755DNAArtificial SequenceSynthesized L1-4NHT (ING1) 607ccatctcctg ccgctctagt aagagtctcn htcatagtaa tggcatcact tattt 5560855DNAArtificial SequenceSynthesized L1-4VAA (ING1) 608ccatctcctg ccgctctagt aagagtctcv aacatagtaa tggcatcact tattt 5560955DNAArtificial SequenceSynthesized L1-4BGG (ING1) 609ccatctcctg ccgctctagt aagagtctcb ggcatagtaa tggcatcact tattt 5561054DNAArtificial SequenceSynthesized L12-1NHT (ING1) 610cgctctagta agagtctcct anhtagtaat ggcatcactt atttgtattg gtat 5461154DNAArtificial SequenceSynthesized L12-1VAA (ING1) 611cgctctagta agagtctcct avaaagtaat ggcatcactt atttgtattg gtat 5461254DNAArtificial SequenceSynthesized L12-1BGG (ING1) 612cgctctagta agagtctcct abggagtaat ggcatcactt atttgtattg gtat

5461354DNAArtificial SequenceSynthesized L12-2NHT (ING1) 613cgctctagta agagtctcct acatnhtaat ggcatcactt atttgtattg gtat 5461454DNAArtificial SequenceSynthesized L12-2VAA (ING1) 614cgctctagta agagtctcct acatvaaaat ggcatcactt atttgtattg gtat 5461554DNAArtificial SequenceSynthesized L12-2BGG (ING1) 615cgctctagta agagtctcct acatbggaat ggcatcactt atttgtattg gtat 5461654DNAArtificial SequenceSynthesized L12-3NHT (ING1) 616cgctctagta agagtctcct acatagtnht ggcatcactt atttgtattg gtat 5461754DNAArtificial SequenceSynthesized L12-3VAA (ING1) 617cgctctagta agagtctcct acatagtvaa ggcatcactt atttgtattg gtat 5461854DNAArtificial SequenceSynthesized L12-3BGG (ING1) 618cgctctagta agagtctcct acatagtbgg ggcatcactt atttgtattg gtat 5461954DNAArtificial SequenceSynthesized L13-1NHT (ING1) 619gagtctccta catagtaatg gcnhtactta tttgtattgg tatttacaga agcc 5462054DNAArtificial SequenceSynthesized L13-1VAA (ING1) 620gagtctccta catagtaatg gcvaaactta tttgtattgg tatttacaga agcc 5462154DNAArtificial SequenceSynthesized L13-1BGG (ING1) 621gagtctccta catagtaatg gcbggactta tttgtattgg tatttacaga agcc 5462254DNAArtificial SequenceSynthesized L13-2NHT (ING1) 622gagtctccta catagtaatg gcatcnhtta tttgtattgg tatttacaga agcc 5462354DNAArtificial SequenceSynthesized L13-2VAA (ING1) 623gagtctccta catagtaatg gcatcvaata tttgtattgg tatttacaga agcc 5462454DNAArtificial SequenceSynthesized L13-2BGG (ING1) 624gagtctccta catagtaatg gcatcbggta tttgtattgg tatttacaga agcc 5462554DNAArtificial SequenceSynthesized L13-3NHT (ING1) 625gagtctccta catagtaatg gcatcactnh tttgtattgg tatttacaga agcc 5462654DNAArtificial SequenceSynthesized L13-3VAA (ING1) 626gagtctccta catagtaatg gcatcactva attgtattgg tatttacaga agcc 5462754DNAArtificial SequenceSynthesized L13-3BGG (ING1) 627gagtctccta catagtaatg gcatcactbg gttgtattgg tatttacaga agcc 5462856DNAArtificial SequenceSynthesized L2-1NHT (ING1) 628cagtctcctc agctcctgat tnhtcagatg tccaaccttg cctcaggagt cccaga 5662956DNAArtificial SequenceSynthesized L2-1VAA (ING1) 629cagtctcctc agctcctgat tvaacagatg tccaaccttg cctcaggagt cccaga 5663056DNAArtificial SequenceSynthesized L2-1BGG (ING1) 630cagtctcctc agctcctgat tbggcagatg tccaaccttg cctcaggagt cccaga 5663156DNAArtificial SequenceSynthesized L2-2NHT (ING1) 631cagtctcctc agctcctgat ttatnhtatg tccaaccttg cctcaggagt cccaga 5663256DNAArtificial SequenceSynthesized L2-2VAA (ING1) 632cagtctcctc agctcctgat ttatvaaatg tccaaccttg cctcaggagt cccaga 5663356DNAArtificial SequenceSynthesized L2-2BGG (ING1) 633cagtctcctc agctcctgat ttatbggatg tccaaccttg cctcaggagt cccaga 5663456DNAArtificial SequenceSynthesized L2-3NHT (ING1) 634cagtctcctc agctcctgat ttatcagnht tccaaccttg cctcaggagt cccaga 5663556DNAArtificial SequenceSynthesized L2-3VAA (ING1) 635cagtctcctc agctcctgat ttatcagvaa tccaaccttg cctcaggagt cccaga 5663656DNAArtificial SequenceSynthesized L2-3BGG (ING1) 636cagtctcctc agctcctgat ttatcagbgg tccaaccttg cctcaggagt cccaga 5663756DNAArtificial SequenceSynthesized L2-4NHT (ING1) 637cagtctcctc agctcctgat ttatcagatg nhtaaccttg cctcaggagt cccaga 5663856DNAArtificial SequenceSynthesized L2-4VAA (ING1) 638cagtctcctc agctcctgat ttatcagatg vaaaaccttg cctcaggagt cccaga 5663956DNAArtificial SequenceSynthesized L2-4BGG (ING1) 639cagtctcctc agctcctgat ttatcagatg bggaaccttg cctcaggagt cccaga 5664056DNAArtificial SequenceSynthesized L2-5NHT (ING1) 640cagtctcctc agctcctgat ttatcagatg tccnhtcttg cctcaggagt cccaga 5664156DNAArtificial SequenceSynthesized L2-5VAA (ING1) 641cagtctcctc agctcctgat ttatcagatg tccvaacttg cctcaggagt cccaga 5664256DNAArtificial SequenceSynthesized L2-5BGG (ING1) 642cagtctcctc agctcctgat ttatcagatg tccbggcttg cctcaggagt cccaga 5664359DNAArtificial SequenceSynthesized H1-1NHT (ING1) 643tcctgcaagg cttctggata tnhtttcaca aaatatggaa tgaactgggt gaagcaggc 5964459DNAArtificial SequenceSynthesized H1-1VAA (ING1) 644tcctgcaagg cttctggata tvaattcaca aaatatggaa tgaactgggt gaagcaggc 5964559DNAArtificial SequenceSynthesized H1-1BGG (ING1) 645tcctgcaagg cttctggata tbggttcaca aaatatggaa tgaactgggt gaagcaggc 5964659DNAArtificial SequenceSynthesized H1-2NHT (ING1) 646tcctgcaagg cttctggata taccttcnht aaatatggaa tgaactgggt gaagcaggc 5964759DNAArtificial SequenceSynthesized H1-2VAA (ING1) 647tcctgcaagg cttctggata taccttcvaa aaatatggaa tgaactgggt gaagcaggc 5964859DNAArtificial SequenceSynthesized H1-2BGG (ING1) 648tcctgcaagg cttctggata taccttcbgg aaatatggaa tgaactgggt gaagcaggc 5964959DNAArtificial SequenceSynthesized H1-3NHT (ING1) 649tcctgcaagg cttctggata taccttcaca nhttatggaa tgaactgggt gaagcaggc 5965059DNAArtificial SequenceSynthesized H1-3VAA (ING1) 650tcctgcaagg cttctggata taccttcaca vaatatggaa tgaactgggt gaagcaggc 5965159DNAArtificial SequenceSynthesized H1-3BGG (ING1) 651tcctgcaagg cttctggata taccttcaca bggtatggaa tgaactgggt gaagcaggc 5965259DNAArtificial SequenceSynthesized H1-4NHT (ING1) 652tcctgcaagg cttctggata taccttcaca aaanhtggaa tgaactgggt gaagcaggc 5965359DNAArtificial SequenceSynthesized H1-4VAA (ING1) 653tcctgcaagg cttctggata taccttcaca aaavaaggaa tgaactgggt gaagcaggc 5965459DNAArtificial SequenceSynthesized H1-4BGG (ING1) 654tcctgcaagg cttctggata taccttcaca aaabggggaa tgaactgggt gaagcaggc 5965559DNAArtificial SequenceSynthesized H1-5NHT (ING1) 655tcctgcaagg cttctggata taccttcaca aaatatnhta tgaactgggt gaagcaggc 5965659DNAArtificial SequenceSynthesized H1-5VAA (ING1) 656tcctgcaagg cttctggata taccttcaca aaatatvaaa tgaactgggt gaagcaggc 5965759DNAArtificial SequenceSynthesized H1-5BGG (ING1) 657tcctgcaagg cttctggata taccttcaca aaatatbgga tgaactgggt gaagcaggc 5965858DNAArtificial SequenceSynthesized H2-1NHT (ING1) 658aagggtttaa agtggatggg cnhtataaac acctacactg aagagcctac atatggtg 5865958DNAArtificial SequenceSynthesized H2-1VAA (ING1) 659aagggtttaa agtggatggg cvaaataaac acctacactg aagagcctac atatggtg 5866058DNAArtificial SequenceSynthesized H2-1BGG (ING1) 660aagggtttaa agtggatggg cbggataaac acctacactg aagagcctac atatggtg 5866158DNAArtificial SequenceSynthesized H2-2NHT (ING1) 661aagggtttaa agtggatggg ctggatanht acctacactg aagagcctac atatggtg 5866258DNAArtificial SequenceSynthesized H2-2VAA (ING1) 662aagggtttaa agtggatggg ctggatavaa acctacactg aagagcctac atatggtg 5866358DNAArtificial SequenceSynthesized H2-2BGG (ING1) 663aagggtttaa agtggatggg ctggatabgg acctacactg aagagcctac atatggtg 5866458DNAArtificial SequenceSynthesized H2-3NHT (ING1) 664aagggtttaa agtggatggg ctggataaac nhttacactg aagagcctac atatggtg 5866558DNAArtificial SequenceSynthesized H2-3VAA (ING1) 665aagggtttaa agtggatggg ctggataaac vaatacactg aagagcctac atatggtg 5866658DNAArtificial SequenceSynthesized H2-3BGG (ING1) 666aagggtttaa agtggatggg ctggataaac bggtacactg aagagcctac atatggtg 5866758DNAArtificial SequenceSynthesized H2-4NHT (ING1) 667aagggtttaa agtggatggg ctggataaac accnhtactg aagagcctac atatggtg 5866858DNAArtificial SequenceSynthesized H2-4VAA (ING1) 668aagggtttaa agtggatggg ctggataaac accvaaactg aagagcctac atatggtg 5866958DNAArtificial SequenceSynthesized H2-4BGG (ING1) 669aagggtttaa agtggatggg ctggataaac accbggactg aagagcctac atatggtg 5867058DNAArtificial SequenceSynthesized H22-1NHT (ING1) 670atgggctgga taaacaccta cnhtgaagag cctacatatg gtgatgactt caagggac 5867158DNAArtificial SequenceSynthesized H22-1VAA (ING1) 671atgggctgga taaacaccta cvaagaagag cctacatatg gtgatgactt caagggac 5867258DNAArtificial SequenceSynthesized H22-1BGG (ING1) 672atgggctgga taaacaccta cbgggaagag cctacatatg gtgatgactt caagggac 5867358DNAArtificial SequenceSynthesized H22-2NHT (ING1) 673atgggctgga taaacaccta cactnhtgag cctacatatg gtgatgactt caagggac 5867458DNAArtificial SequenceSynthesized H22-2VAA (ING1) 674atgggctgga taaacaccta cactvaagag cctacatatg gtgatgactt caagggac 5867558DNAArtificial SequenceSynthesized H22-2BGG (ING1) 675atgggctgga taaacaccta cactbgggag cctacatatg gtgatgactt caagggac 5867658DNAArtificial SequenceSynthesized H22-3NHT (ING1) 676atgggctgga taaacaccta cactgaanht cctacatatg gtgatgactt caagggac 5867758DNAArtificial SequenceSynthesized H22-3VAA (ING1) 677atgggctgga taaacaccta cactgaavaa cctacatatg gtgatgactt caagggac 5867858DNAArtificial SequenceSynthesized H22-3BGG (ING1) 678atgggctgga taaacaccta cactgaabgg cctacatatg gtgatgactt caagggac 5867958DNAArtificial SequenceSynthesized H22-4NHT (ING1) 679atgggctgga taaacaccta cactgaagag nhtacatatg gtgatgactt caagggac 5868058DNAArtificial SequenceSynthesized H22-4VAA (ING1) 680atgggctgga taaacaccta cactgaagag vaaacatatg gtgatgactt caagggac 5868158DNAArtificial SequenceSynthesized H22-4BGG (ING1) 681atgggctgga taaacaccta cactgaagag bggacatatg gtgatgactt caagggac 5868258DNAArtificial SequenceSynthesized H22-5NHT (ING1) 682atgggctgga taaacaccta cactgaagag cctnhttatg gtgatgactt caagggac 5868358DNAArtificial SequenceSynthesized H22-5VAA (ING1) 683atgggctgga taaacaccta cactgaagag cctvaatatg gtgatgactt caagggac 5868458DNAArtificial SequenceSynthesized H22-5BGG (ING1) 684atgggctgga taaacaccta cactgaagag cctbggtatg gtgatgactt caagggac 5868553DNAArtificial SequenceSynthesized H3-1NHT (ING1) 685gctacatatt tctgtgcaag atttnhttct gctgtggact actggggtca agg 5368653DNAArtificial SequenceSynthesized H3-1VAA (ING1) 686gctacatatt tctgtgcaag atttvaatct gctgtggact actggggtca agg 5368753DNAArtificial SequenceSynthesized H3-1BGG (ING1) 687gctacatatt tctgtgcaag atttbggtct gctgtggact actggggtca agg 5368853DNAArtificial SequenceSynthesized H3-2NHT (ING1) 688gctacatatt tctgtgcaag atttggcnht gctgtggact actggggtca agg 5368953DNAArtificial SequenceSynthesized H3-2VAA (ING1) 689gctacatatt tctgtgcaag atttggcvaa gctgtggact actggggtca agg 5369053DNAArtificial SequenceSynthesized H3-2BGG (ING1) 690gctacatatt tctgtgcaag atttggcbgg gctgtggact actggggtca agg 5369153DNAArtificial SequenceSynthesized H3-3NHT (ING1) 691gctacatatt tctgtgcaag atttggctct nhtgtggact actggggtca agg 5369253DNAArtificial SequenceSynthesized H3-3VAA (ING1) 692gctacatatt tctgtgcaag atttggctct vaagtggact actggggtca agg 5369353DNAArtificial SequenceSynthesized H3-3BGG (ING1) 693gctacatatt tctgtgcaag atttggctct bgggtggact actggggtca agg 5369446DNAArtificial SequenceSynthesized H32-1NHT (ING1) 694gcaagatttg gctctgctgt gnhttactgg ggtcaaggaa cctcgg 4669546DNAArtificial SequenceSynthesized H32-1VAA (ING1) 695gcaagatttg gctctgctgt gvaatactgg ggtcaaggaa cctcgg 4669646DNAArtificial SequenceSynthesized H32-1BGG (ING1) 696gcaagatttg gctctgctgt gbggtactgg ggtcaaggaa cctcgg 4669746DNAArtificial SequenceSynthesized H32-2NHT (ING1) 697gcaagatttg gctctgctgt ggacnhttgg ggtcaaggaa cctcgg 4669846DNAArtificial SequenceSynthesized H32-2VAA (ING1) 698gcaagatttg gctctgctgt ggacvaatgg ggtcaaggaa cctcgg 4669946DNAArtificial SequenceSynthesized H32-2BGG (ING1) 699gcaagatttg gctctgctgt ggacbggtgg ggtcaaggaa cctcgg 4670054DNAArtificial SequenceSynthesized H1-1NHT (IL1B) primer 700cttgcgctgc ttccggattc nhtttctcta agtactttat gttttgggtt cgcc 5470154DNAArtificial SequenceSynthesized H1-1VAA (IL1B) primer 701cttgcgctgc ttccggattc vaattctcta agtactttat gttttgggtt cgcc 5470254DNAArtificial SequenceSynthesized H1-1BGG (IL1B) primer 702cttgcgctgc ttccggattc bggttctcta agtactttat gttttgggtt cgcc 5470354DNAArtificial SequenceSynthesized H1-2NHT (IL1B) primer 703cttgcgctgc ttccggattc actttcnhta agtactttat gttttgggtt cgcc 5470454DNAArtificial SequenceSynthesized H1-2VAA (IL1B) primer 704cttgcgctgc ttccggattc actttcvaaa agtactttat gttttgggtt cgcc 5470554DNAArtificial SequenceSynthesized H1-2BGG (IL1B) primer 705cttgcgctgc ttccggattc actttcbgga agtactttat gttttgggtt cgcc 5470654DNAArtificial SequenceSynthesized H1-3NHT (IL1B) primer 706cttgcgctgc ttccggattc actttctctn httactttat gttttgggtt cgcc 5470754DNAArtificial SequenceSynthesized H1-3VAA (IL1B) primer 707cttgcgctgc ttccggattc actttctctv aatactttat gttttgggtt cgcc 5470854DNAArtificial SequenceSynthesized H1-3BGG (IL1B) primer 708cttgcgctgc ttccggattc actttctctb ggtactttat gttttgggtt cgcc 5470953DNAArtificial SequenceSynthesized H12-1NHT (IL1B) primer 709ttccggattc actttctcta agnhttttat gttttgggtt cgccaagctc ctg 5371053DNAArtificial SequenceSynthesized H12-1VAA (IL1B) primer 710ttccggattc actttctcta agvaatttat gttttgggtt cgccaagctc ctg 5371153DNAArtificial SequenceSynthesized H12-1BGG (IL1B) primer 711ttccggattc actttctcta agbggtttat gttttgggtt cgccaagctc ctg 5371253DNAArtificial SequenceSynthesized H12-2NHT (IL1B) primer 712ttccggattc actttctcta agtacnhtat gttttgggtt cgccaagctc ctg 5371353DNAArtificial SequenceSynthesized H12-2VAA (IL1B) primer 713ttccggattc actttctcta agtacvaaat gttttgggtt cgccaagctc ctg 5371453DNAArtificial SequenceSynthesized H12-2BGG (IL1B) primer 714ttccggattc actttctcta agtacbggat gttttgggtt cgccaagctc ctg 5371553DNAArtificial SequenceSynthesized H12-3NHT (IL1B) primer 715ttccggattc actttctcta agtactttat gnhttgggtt cgccaagctc ctg 5371653DNAArtificial SequenceSynthesized H12-3VAA (IL1B) primer 716ttccggattc actttctcta agtactttat gvaatgggtt cgccaagctc ctg 5371753DNAArtificial SequenceSynthesized H12-3BGG (IL1B) primer 717ttccggattc actttctcta agtactttat gbggtgggtt cgccaagctc ctg 5371857DNAArtificial SequenceSynthesized H2-1NHT (IL1B) primer 718taaaggtttg gagtgggttt ctnhtatctc tccttctggt ggcatgactc gttatgc 5771957DNAArtificial SequenceSynthesized H2-1VAA (IL1B) primer 719taaaggtttg gagtgggttt ctvaaatctc tccttctggt ggcatgactc gttatgc 5772057DNAArtificial SequenceSynthesized H2-1BGG (IL1B) primer 720taaaggtttg gagtgggttt ctbggatctc tccttctggt ggcatgactc gttatgc 5772157DNAArtificial SequenceSynthesized H2-2NHT (IL1B) primer 721taaaggtttg gagtgggttt ctgttnhttc tccttctggt ggcatgactc gttatgc 5772257DNAArtificial SequenceSynthesized H2-2VAA (IL1B) primer 722taaaggtttg gagtgggttt ctgttvaatc tccttctggt ggcatgactc gttatgc 5772357DNAArtificial SequenceSynthesized H2-2BGG (IL1B) primer 723taaaggtttg gagtgggttt ctgttbggtc tccttctggt ggcatgactc gttatgc 5772457DNAArtificial SequenceSynthesized H2-3NHT (IL1B) primer 724taaaggtttg gagtgggttt ctgttatcnh tccttctggt ggcatgactc gttatgc 5772557DNAArtificial SequenceSynthesized H2-3VAA (IL1B) primer 725taaaggtttg gagtgggttt ctgttatcva accttctggt ggcatgactc gttatgc 5772657DNAArtificial SequenceSynthesized H2-3BGG (IL1B) primer 726taaaggtttg gagtgggttt ctgttatcbg gccttctggt ggcatgactc gttatgc 5772757DNAArtificial SequenceSynthesized H2-4NHT (IL1B) primer 727taaaggtttg gagtgggttt ctgttatctc tnhttctggt ggcatgactc gttatgc 5772857DNAArtificial SequenceSynthesized H2-4VAA (IL1B) primer 728taaaggtttg gagtgggttt ctgttatctc tvaatctggt ggcatgactc gttatgc 5772957DNAArtificial SequenceSynthesized H2-4BGG (IL1B) primer 729taaaggtttg gagtgggttt ctgttatctc tbggtctggt ggcatgactc gttatgc 5773057DNAArtificial SequenceSynthesized H2-5NHT (IL1B) primer 730taaaggtttg gagtgggttt ctgttatctc tcctnhtggt ggcatgactc gttatgc 5773157DNAArtificial SequenceSynthesized H2-5VAA (IL1B) primer 731taaaggtttg gagtgggttt ctgttatctc tcctvaaggt ggcatgactc gttatgc 5773257DNAArtificial SequenceSynthesized H2-5BGG (IL1B) primer 732taaaggtttg gagtgggttt ctgttatctc tcctbggggt ggcatgactc gttatgc 5773359DNAArtificial SequenceSynthesized H22-1NHT (IL1B) primer 733ggtttctgtt atctctcctt ctnhtggcat gactcgttat gctgactccg ttaaaggtc 5973459DNAArtificial SequenceSynthesized H22-1VAA (IL1B) primer 734ggtttctgtt atctctcctt ctvaaggcat gactcgttat gctgactccg ttaaaggtc 5973559DNAArtificial SequenceSynthesized H22-1BGG (IL1B) primer 735ggtttctgtt atctctcctt ctbggggcat gactcgttat gctgactccg ttaaaggtc 5973659DNAArtificial SequenceSynthesized H22-2NHT (IL1B) primer 736ggtttctgtt atctctcctt ctggtnhtat gactcgttat gctgactccg ttaaaggtc 5973759DNAArtificial SequenceSynthesized H22-2VAA (IL1B) primer 737ggtttctgtt atctctcctt ctggtvaaat gactcgttat gctgactccg ttaaaggtc 5973859DNAArtificial SequenceSynthesized H22-2BGG (IL1B) primer

738ggtttctgtt atctctcctt ctggtbggat gactcgttat gctgactccg ttaaaggtc 5973959DNAArtificial SequenceSynthesized H22-3NHT (IL1B) primer 739ggtttctgtt atctctcctt ctggtggcnh tactcgttat gctgactccg ttaaaggtc 5974059DNAArtificial SequenceSynthesized H22-3VAA (IL1B) primer 740ggtttctgtt atctctcctt ctggtggcva aactcgttat gctgactccg ttaaaggtc 5974159DNAArtificial SequenceSynthesized H22-3BGG (IL1B) primer 741ggtttctgtt atctctcctt ctggtggcbg gactcgttat gctgactccg ttaaaggtc 5974259DNAArtificial SequenceSynthesized H22-4NHT (IL1B) primer 742ggtttctgtt atctctcctt ctggtggcat gnhtcgttat gctgactccg ttaaaggtc 5974359DNAArtificial SequenceSynthesized H22-4VAA (IL1B) primer 743ggtttctgtt atctctcctt ctggtggcat gvaacgttat gctgactccg ttaaaggtc 5974459DNAArtificial SequenceSynthesized H22-4BGG (IL1B) primer 744ggtttctgtt atctctcctt ctggtggcat gbggcgttat gctgactccg ttaaaggtc 5974559DNAArtificial SequenceSynthesized H22-5NHT (IL1B) primer 745ggtttctgtt atctctcctt ctggtggcat gactnhttat gctgactccg ttaaaggtc 5974659DNAArtificial SequenceSynthesized H22-5VAA (IL1B) primer 746ggtttctgtt atctctcctt ctggtggcat gactvaatat gctgactccg ttaaaggtc 5974759DNAArtificial SequenceSynthesized H22-5BGG (IL1B) primer 747ggtttctgtt atctctcctt ctggtggcat gactbggtat gctgactccg ttaaaggtc 5974856DNAArtificial SequenceSynthesized H3-1NHT (IL1B) primer 748gcagtctact attgtgcgag anhtggctac ggtggtaact ctgactactg gggcca 5674956DNAArtificial SequenceSynthesized H3-1VAA (IL1B) primer 749gcagtctact attgtgcgag avaaggctac ggtggtaact ctgactactg gggcca 5675056DNAArtificial SequenceSynthesized H3-1BGG (IL1B) primer 750gcagtctact attgtgcgag abggggctac ggtggtaact ctgactactg gggcca 5675156DNAArtificial SequenceSynthesized H3-2NHT (IL1B) primer 751gcagtctact attgtgcgag agtcnhttac ggtggtaact ctgactactg gggcca 5675256DNAArtificial SequenceSynthesized H3-2VAA (IL1B) primer 752gcagtctact attgtgcgag agtcvaatac ggtggtaact ctgactactg gggcca 5675356DNAArtificial SequenceSynthesized H3-2BGG (IL1B) primer 753gcagtctact attgtgcgag agtcbggtac ggtggtaact ctgactactg gggcca 5675456DNAArtificial SequenceSynthesized H3-3NHT (IL1B) primer 754gcagtctact attgtgcgag agtcggcnht ggtggtaact ctgactactg gggcca 5675556DNAArtificial SequenceSynthesized H3-3VAA (IL1B) primer 755gcagtctact attgtgcgag agtcggcvaa ggtggtaact ctgactactg gggcca 5675656DNAArtificial SequenceSynthesized H3-3BGG (IL1B) primer 756gcagtctact attgtgcgag agtcggcbgg ggtggtaact ctgactactg gggcca 5675756DNAArtificial SequenceSynthesized H3-4NHT (IL1B) primer 757gcagtctact attgtgcgag agtcggctac nhtggtaact ctgactactg gggcca 5675856DNAArtificial SequenceSynthesized H3-4VAA (IL1B) primer 758gcagtctact attgtgcgag agtcggctac vaaggtaact ctgactactg gggcca 5675956DNAArtificial SequenceSynthesized H3-4BGG (IL1B) primer 759gcagtctact attgtgcgag agtcggctac bggggtaact ctgactactg gggcca 5676056DNAArtificial SequenceSynthesized H3-5NHT (IL1B) primer 760gcagtctact attgtgcgag agtcggctac ggtnhtaact ctgactactg gggcca 5676156DNAArtificial SequenceSynthesized H3-5VAA (IL1B) primer 761gcagtctact attgtgcgag agtcggctac ggtvaaaact ctgactactg gggcca 5676256DNAArtificial SequenceSynthesized H3-5BGG (IL1B) primer 762gcagtctact attgtgcgag agtcggctac ggtbggaact ctgactactg gggcca 5676349DNAArtificial SequenceSynthesized H32-1NHT (IL1B) primer 763gagagtcggc tacggtggtn httctgacta ctggggccag ggaaccctg 4976449DNAArtificial SequenceSynthesized H32-1VAA (IL1B) primer 764gagagtcggc tacggtggtv aatctgacta ctggggccag ggaaccctg 4976549DNAArtificial SequenceSynthesized H32-1BGG (IL1B) primer 765gagagtcggc tacggtggtb ggtctgacta ctggggccag ggaaccctg 4976649DNAArtificial SequenceSynthesized H32-2NHT (IL1B) primer 766gagagtcggc tacggtggta acnhtgacta ctggggccag ggaaccctg 4976749DNAArtificial SequenceSynthesized H32-2VAA (IL1B) primer 767gagagtcggc tacggtggta acvaagacta ctggggccag ggaaccctg 4976849DNAArtificial SequenceSynthesized H32-2BGG (IL1B) primer 768gagagtcggc tacggtggta acbgggacta ctggggccag ggaaccctg 4976949DNAArtificial SequenceSynthesized H32-3VAA (IL1B) primer 769gagagtcggc tacggtggta actctvaata ctggggccag ggaaccctg 4977049DNAArtificial SequenceSynthesized H32-3BGG (IL1B) primer 770gagagtcggc tacggtggta actctbggta ctggggccag ggaaccctg 4977149DNAArtificial SequenceSynthesized H32-4NHT (IL1B) primer 771gagagtcggc tacggtggta actctgacnh ttggggccag ggaaccctg 4977249DNAArtificial SequenceSynthesized H32-4VAA (IL1B) primer 772gagagtcggc tacggtggta actctgacva atggggccag ggaaccctg 4977349DNAArtificial SequenceSynthesized H32-4BGG (IL1B) primer 773gagagtcggc tacggtggta actctgacbg gtggggccag ggaaccctg 4977458DNAArtificial SequenceSynthesized L1-1NHT (IL1B) primer 774ccatcatttg tcgggcgagt nhtgatatta acaggtggtt agcctggtat cagcagac 5877558DNAArtificial SequenceSynthesized L1-1VAA (IL1B) primer 775ccatcatttg tcgggcgagt vaagatatta acaggtggtt agcctggtat cagcagac 5877658DNAArtificial SequenceSynthesized L1-1BGG (IL1B) primer 776ccatcatttg tcgggcgagt bgggatatta acaggtggtt agcctggtat cagcagac 5877758DNAArtificial SequenceSynthesized L1-2NHT (IL1B) primer 777ccatcatttg tcgggcgagt cagnhtatta acaggtggtt agcctggtat cagcagac 5877858DNAArtificial SequenceSynthesized L1-2VAA (IL1B) primer 778ccatcatttg tcgggcgagt cagvaaatta acaggtggtt agcctggtat cagcagac 5877958DNAArtificial SequenceSynthesized L1-2BGG (IL1B) primer 779ccatcatttg tcgggcgagt cagbggatta acaggtggtt agcctggtat cagcagac 5878058DNAArtificial SequenceSynthesized L1-3NHT (IL1B) primer 780ccatcatttg tcgggcgagt caggatattn htaggtggtt agcctggtat cagcagac 5878158DNAArtificial SequenceSynthesized L1-3VAA (IL1B) primer 781ccatcatttg tcgggcgagt caggatattv aaaggtggtt agcctggtat cagcagac 5878258DNAArtificial SequenceSynthesized L1-3BGG (IL1B) primer 782ccatcatttg tcgggcgagt caggatattb ggaggtggtt agcctggtat cagcagac 5878358DNAArtificial SequenceSynthesized L1-4NHT (IL1B) primer 783ccatcatttg tcgggcgagt caggatatta acnhttggtt agcctggtat cagcagac 5878458DNAArtificial SequenceSynthesized L1-4VAA (IL1B) primer 784ccatcatttg tcgggcgagt caggatatta acvaatggtt agcctggtat cagcagac 5878558DNAArtificial SequenceSynthesized L1-4BGG (IL1B) primer 785ccatcatttg tcgggcgagt caggatatta acbggtggtt agcctggtat cagcagac 5878658DNAArtificial SequenceSynthesized L1-5NHT (IL1B) primer 786ccatcatttg tcgggcgagt caggatatta acaggnhttt agcctggtat cagcagac 5878758DNAArtificial SequenceSynthesized L1-5VAA (IL1B) primer 787ccatcatttg tcgggcgagt caggatatta acaggvaatt agcctggtat cagcagac 5878855DNAArtificial SequenceSynthesized L1-5BGG (IL1B) primer 788atgcccctaa gctcctgatc cattctgcaa ccbggctgca aagtggggtc ccatc 5578957DNAArtificial SequenceSynthesized L3-1NHT (IL1B) primer 789gcaacttact attgtcagca gnhtgacagt ttcccgctca ctttcggcgg agggacc 5779057DNAArtificial SequenceSynthesized L3-1VAA (IL1B) primer 790gcaacttact attgtcagca gvaagacagt ttcccgctca ctttcggcgg agggacc 5779157DNAArtificial SequenceSynthesized L3-1BGG (IL1B) primer 791gcaacttact attgtcagca gbgggacagt ttcccgctca ctttcggcgg agggacc 5779257DNAArtificial SequenceSynthesized L3-2NHT (IL1B) primer 792gcaacttact attgtcagca ggctnhtagt ttcccgctca ctttcggcgg agggacc 5779357DNAArtificial SequenceSynthesized L3-2VAA (IL1B) primer 793gcaacttact attgtcagca ggctvaaagt ttcccgctca ctttcggcgg agggacc 5779457DNAArtificial SequenceSynthesized L3-2BGG (IL1B) primer 794gcaacttact attgtcagca ggctbggagt ttcccgctca ctttcggcgg agggacc 5779557DNAArtificial SequenceSynthesized L3-3NHT (IL1B) primer 795gcaacttact attgtcagca ggctgacnht ttcccgctca ctttcggcgg agggacc 5779657DNAArtificial SequenceSynthesized L3-3VAA (IL1B) primer 796gcaacttact attgtcagca ggctgacvaa ttcccgctca ctttcggcgg agggacc 5779757DNAArtificial SequenceSynthesized L3-3BGG (IL1B) primer 797gcaacttact attgtcagca ggctgacbgg ttcccgctca ctttcggcgg agggacc 5779857DNAArtificial SequenceSynthesized L3-4NHT (IL1B) primer 798gcaacttact attgtcagca ggctgacagt nhtccgctca ctttcggcgg agggacc 5779957DNAArtificial SequenceSynthesized L3-4VAA (IL1B) primer 799gcaacttact attgtcagca ggctgacagt vaaccgctca ctttcggcgg agggacc 5780057DNAArtificial SequenceSynthesized L3-4BGG (IL1B) primer 800gcaacttact attgtcagca ggctgacagt bggccgctca ctttcggcgg agggacc 5780157DNAArtificial SequenceSynthesized L3-5NHT (IL1B) primer 801gcaacttact attgtcagca ggctgacagt ttcnhtctca ctttcggcgg agggacc 5780257DNAArtificial SequenceSynthesized L3-5VAA (IL1B) primer 802gcaacttact attgtcagca ggctgacagt ttcvaactca ctttcggcgg agggacc 5780357DNAArtificial SequenceSynthesized L3-5BGG (IL1B) primer 803gcaacttact attgtcagca ggctgacagt ttcbggctca ctttcggcgg agggacc 5780457DNAArtificial SequenceSynthesized L3-6NHT (IL1B) primer 804gcaacttact attgtcagca ggctgacagt ttcccgnhta ctttcggcgg agggacc 5780557DNAArtificial SequenceSynthesized L3-6VAA (IL1B) primer 805gcaacttact attgtcagca ggctgacagt ttcccgvaaa ctttcggcgg agggacc 5780657DNAArtificial SequenceSynthesized L3-6BGG (IL1B) primer 806gcaacttact attgtcagca ggctgacagt ttcccgbgga ctttcggcgg agggacc 5780759DNAArtificial SequenceSynthesized L3-E98T 807ggtgtttatt actgtgctca aaatctaact cttcctcgga cgttcggtgg aggcaccaa 5980856DNAArtificial SequenceSynthesized L2-Q55R 808cagtctcctc agctcctgat ttatcgaatg tccaaccttg cctcaggagt cccaga 5680959DNAArtificial SequenceSynthesized H1-G33F 809tcctgcaagg cttctggata taccttcaca aaatatttca tgaactgggt gaagcaggc 5981058DNAArtificial SequenceSynthesized H2-T53I 810aagggtttaa agtggatggg ctggataaac atctacactg aagagcctac atatggtg 5881153DNAArtificial SequenceSynthesized H3-G100R 811gctacatatt tctgtgcaag atttcgttct gctgtggact actggggtca agg 5381240DNAArtificial SequenceSynthesized AscIR 812atatatggcg cgccttatta acactctccc ctgttgaagc 4081321DNAArtificial SequenceSynthesized AscIF 813taataaggcg cgcctaacca t 2181420DNAArtificial SequenceSynthesized HindIII-F 814ttacgccaag ctttggagcc 20815112PRTArtificial SequenceSynthesized murING1 (light chain) 815Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly1 5 10 15Thr Ser Gly Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110816115PRTArtificial SequenceSynthesized mK2 (light chain) 816Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110Lys Arg Ala 115817116PRTArtificial SequenceSynthesized murING1 (heavy chain) 817Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Glu Glu Pro Thr Tyr Gly Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Asn65 70 75 80Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Phe Gly Ser Ala Val Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser 115818129PRTArtificial SequenceSynthesized mH2a (heavy chain) 818Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Asn Trp Val Lys Gln Ser Pro Gly Lys Ser Leu Glu Trp 35 40 45Ile Gly Asp Ile Asn Pro Gly Asn Gly Gly Thr Ser Tyr Asn Gln Lys 50 55 60Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala65 70 75 80Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95Cys Ala Arg Xaa Xaa Tyr Tyr Ser Ser Ser Xaa Met Xaa Ala Xaa Xaa 100 105 110Tyr Tyr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125Ser819113PRTArtificial SequenceSynthesized hK1 819Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Leu Val Ser Ile 20 25 30Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser65 70 75 80Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 85 90 95Leu Pro Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110Thr 820113PRTArtificial SequenceSynthesized hK2 820Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asp Gly Asn Asn Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110Thr 821111PRTArtificial SequenceSynthesized hK3 821Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110822114PRTArtificial SequenceSynthesized hK4 822Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Thr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110Arg Thr 823115PRTArtificial SequenceSynthesized mK1 823Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110Lys Arg Ala 115824115PRTArtificial SequenceSynthesized mK2 824Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110Lys Arg Ala 115825114PRTArtificial SequenceSynthesized mK3 825Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Ala 826112PRTArtificial SequenceSynthesized mK4 826Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 20 25 30Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Xaa Ser Pro Lys Leu Trp 35 40 45Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu65 70 75 80Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95Xaa Gly Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 110827111PRTArtificial SequenceSynthesized mK5 827Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Asp Ile Ser Asn 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ser Pro Lys Leu Leu 35 40 45Ile Tyr Tyr Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu65 70 75 80Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 110828111PRTArtificial SequenceSynthesized mK6 828Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Met 85 90 95Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala 100 105 110829118PRTArtificial SequenceSynthesized hL1 829Glx Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Ile Gly Asn 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Xaa Gly Leu65 70 75 80Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser 85 90 95Leu Ser Ala Xaa Asn Ser Ala Pro Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro 115830114PRTArtificial SequenceSynthesized hL2 830Glx Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Ala Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Leu Ile Tyr Asp Val Thr Asp Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Gly Gly Gly 85 90 95Ser Xaa Xaa Asn Val Phe Gly Gly Gly Thr Lys Xaa Thr Val Leu Gly 100 105 110Gln Pro 831113PRTArtificial SequenceSynthesized hL3 831Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Ser Gly Asp Asn Leu Gly Asp Lys Tyr Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Asp Asp Xaa Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Gln Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Ser Asp His 85 90 95Pro Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110Pro 832112PRTArtificial SequenceSynthesized hL4 832Ser Glu Leu Thr Gln Pro Pro Ser Xaa Val Ser Val Ala Xaa Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Xaa Gly Asp Ser Asn Leu Gly Xaa Tyr Asp 20 25 30Ala Ser Trp Tyr Gln Gln Lys Pro Xaa Gln Ala Pro Xaa Leu Val Ile 35 40 45Tyr Gly Xaa Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Ser Ser Gly Xaa Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala65 70 75 80Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Xaa 85 90 95Xaa Xaa Xaa Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110833113PRTArtificial SequenceSynthesized hL5 833Xaa Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1 5 10 15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Ala Gly Lys Ala Pro Lys Val 35 40 45Ile Ile Tyr Glu Val Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Glu Gly Ser 85 90 95Asp Asn Phe Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110Pro 834111PRTArtificial SequenceSynthesized hL6 834Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys1 5 10 15Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu Lys Thr65 70 75 80Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Asn Asn His 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110835116PRTArtificial SequenceSynthesized mL 835Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu1 5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75 80Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95His Glu Gln Phe Val Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110Leu Gly Gln Pro 115836129PRTArtificial SequenceSynthesized hH1 836Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Tyr Gly Asn Gly Asp Thr Asn Tyr Ala Gln Lys 50 55 60Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala65 70 75 80Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Ala Pro Gly Tyr Gly Ser Gly Gly Gly Cys Tyr Arg Gly 100 105 110Asp Tyr Xaa Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125Ser837132PRTArtificial SequenceSynthesized hH2 837Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Tyr 20 25 30Xaa Trp Ser Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Arg Ile Tyr Tyr Arg Ala Tyr Ser Gly Ser Thr Xaa Tyr 50 55 60Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys65 70 75 80Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 85 90 95Val Tyr Tyr Cys Ala Arg Glu Xaa Xaa Xaa Gly Xaa Xaa Gly Asp Asp 100 105 110Tyr Tyr Tyr Xaa Xaa Gly Phe Asp Val Trp Gly Gln Gly Thr Leu Val 115 120 125Thr Val Ser Ser 130838130PRTArtificial SequenceSynthesized hH3 838Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Gly Lys Thr Asp Gly Gly Ser Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Leu Ser Gly Xaa Tyr Tyr 100 105 110Tyr Tyr His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 125Ser Ser 130839130PRTArtificial SequenceSynthesized mH1a 839Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30Tyr Trp Asn Asn Ser Trp Ile Arg Xaa Phe Pro Gly Asn Lys Leu Glu 35 40 45Trp Met Gly Tyr Ile Ser Xaa Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro 50 55 60Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln65 70 75 80Tyr Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 85 90 95Tyr Cys Ala Arg Xaa Xaa Tyr Gly Tyr Xaa Xaa Tyr Xaa Tyr Asp Xaa 100 105 110Tyr Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 125Ser Ser 130840129PRTArtificial SequenceSynthesized mH1b 840Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Leu Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala 50 55 60Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val65 70 75 80Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr 85 90 95Cys Ala Arg Asp Arg Gly Xaa Tyr Tyr Tyr Xaa Ser Gly Xaa Xaa Xaa 100 105 110Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 115 120 125Ser841129PRTArtificial SequenceSynthesized mH2a 841Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Asn Trp Val Lys Gln Ser Pro Gly Lys Ser Leu Glu Trp 35 40 45Ile Gly Asp Ile Asn Pro Gly Asn Gly Gly Thr Ser Tyr Asn Gln Lys 50 55 60Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala65 70 75 80Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95Cys Ala Arg Xaa Xaa Tyr Tyr Ser Ser Ser Xaa Met Xaa Ala Xaa Xaa 100 105 110Tyr Tyr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125Ser842127PRTArtificial SequenceSynthesized mH2b 842Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75

80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Tyr Tyr Gly Gly Ser Ser Xaa Xaa Val Tyr Xaa Tyr Trp 100 105 110Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125843125PRTArtificial SequenceSynthesized mH2c 843Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Tyr Tyr Tyr Tyr Asp Ser Xaa Val Gly Tyr Tyr Ala Met 100 105 110Asp Tyr Trp Gly Gln Gly Thr Xaa Val Thr Val Ser Ser 115 120 125844130PRTArtificial SequenceSynthesized mH3a 844Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Ile 35 40 45Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Thr Ser Gln Ser Ile65 70 75 80Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95Tyr Cys Ala Arg Asp Tyr Tyr Tyr Gly Ser Ser Tyr Tyr Glu Gly Pro 100 105 110Val Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val 115 120 125Ser Ser 130845127PRTArtificial SequenceSynthesized mH3b 845Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Lys Ala Asp Ser Ser Thr Ile Asn Tyr Thr Pro 50 55 60Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr 85 90 95Tyr Cys Ala Arg Leu Gly Gly Tyr Gly Tyr Phe Gly Ser Ser Tyr Tyr 100 105 110Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125846127PRTArtificial SequenceSynthesized mH3c 846Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Trp Met Asn Xaa Xaa Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu 35 40 45Trp Val Ala Glu Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr 50 55 60Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys65 70 75 80Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly 85 90 95Ile Tyr Tyr Cys Thr Thr Gly Gly Tyr Gly Gly Xaa Arg Arg Ser Xaa 100 105 110Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125847129PRTArtificial SequenceSynthesized mH3d 847Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Lys Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Ala Arg Gly Xaa Tyr Tyr Tyr Xaa Xaa Gly Ser Ala Pro Phe 100 105 110Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 115 120 125Ser848128PRTArtificial SequenceSynthesized mH5a 848Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ala Gly Ser1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Gly Asn Gly Tyr Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Ser Xaa Tyr Tyr Gly Gly Ser Tyr Tyr Tyr Xaa Phe Ala Tyr 100 105 110Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 125849114PRTArtificial SequenceSynthesized mH5b 849Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Ala Gly Ser1 5 10 15Ser Val Lys Met Ser Cys Ser Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 20 25 30Gly Leu Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Xaa Xaa 35 40 45Gly Tyr Ile Ser Ser Ser Ser Ala Tyr Pro Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Val Arg Val Ile Ser Arg Tyr Phe Asp Gly Trp Gly Gln Gly Thr 100 105 110Leu Val 850132PRTArtificial SequenceSynthesized mHms 850Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Asn Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Gly Trp Ile Asn Ser Lys Leu Gly Gly Gly Ala Ile Tyr Tyr 50 55 60Ala Asp Thr Xaa Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys65 70 75 80Ser Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala 85 90 95Thr Tyr Tyr Cys Ala Arg Xaa Gly Tyr Tyr Gly Gly Arg Arg Ser Xaa 100 105 110Xaa Xaa Ser Tyr Trp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val 115 120 125Thr Val Ser Ser 13085163DNAArtificial SequenceSynthesized XPA CDR1 coding sequence 851tctttcttgc gctgcttccg gattcacttt ctctaagtac tttatgtttt gggttcgcca 60agc 6385263DNAArtificial SequenceSynthesized XPA CDR1 coding sequence (antisense) 852agaaagaacg cgacgaaggc ctaagtgaaa gagattcatg aaatacaaaa cccaagcggt 60tcg 6385347DNAArtificial SequenceSynthesized primer 1 for mutation of G position in XPA CDR1 853gtctttcttg cgctgcttcc nhtttcactt tctctaagta ctttatg 4785447DNAArtificial SequenceSynthesized primer 1 for mutation of G position in XPA CDR1 (antisense) 854cagaaagaac gcgacgaagg ndaaagtgaa agagattcat gaaatac 4785547DNAArtificial SequenceSynthesized primer 2 for mutation of G position in XPA CDR1 855gtctttcttg cgctgcttcc vaattcactt tctctaagta ctttatg 4785647DNAArtificial SequenceSynthesized primer 2 for mutation of G position in XPA CDR1(antisense) 856cagaaagaac gcgacgaagg bttaagtgaa agagattcat gaaatac 4785747DNAArtificial SequenceSynthesized primer 3 for mutation of G position in XPA CDR1 857gtctttcttg cgctgcttcc bggttcactt tctctaagta ctttatg 4785847DNAArtificial SequenceSynthesized primer 3 for mutation of G position in XPA CDR1 (antisense) 858cagaaagaac gcgacgaagg vccaagtgaa agagattcat gaaatac 4785945DNAArtificial SequenceSynthesized primer 1 for mutation of F position in XPA CDR1 859ctttcttgcg ctgcttccgg anhtactttc tctaagtact ttatg 4586045DNAArtificial SequenceSynthesized primer 1 for mutation of F position in XPA CDR1 (antisense) 860gaaagaacgc gacgaaggcc tndatgaaag agattcatga aatac 4586145DNAArtificial SequenceSynthesized primer 2 for mutation of F position in XPA CDR1 861ctttcttgcg ctgcttccgg avaaactttc tctaagtact ttatg 4586245DNAArtificial SequenceSynthesized primer 2 for mutation of F position in XPA CDR1 (antisense) 862gaaagaacgc gacgaaggcc tbtttgaaag agattcatga aatac 4586345DNAArtificial SequenceSynthesized primer 3 for mutation of F position in XPA CDR1 863ctttcttgcg ctgcttccgg abggactttc tctaagtact ttatg 4586445DNAArtificial SequenceSynthesized primer 3 for mutation of F position in XPA CDR1 (antisense) 864gaaagaacgc gacgaaggcc tvcctgaaag agattcatga aatac 4586546DNAArtificial SequenceSynthesized primer 1 for mutation of T position in XPA CDR1 865cttgcgctgc ttccggattc nhtttctcta agtactttat gttttg 4686646DNAArtificial SequenceSynthesized primer 1 for mutation of T position in XPA CDR1 (antisense) 866gaacgcgacg aaggcctaag ndaaagagat tcatgaaata caaaac 4686746DNAArtificial SequenceSynthesized primer 2 for mutation of T position in XPA CDR1 867cttgcgctgc ttccggattc vaattctcta agtactttat gttttg 4686846DNAArtificial SequenceSynthesized primer 2 for mutation of T position in XPA CDR1 (antisense) 868gaacgcgacg aaggcctaag bttaagagat tcatgaaata caaaac 4686946DNAArtificial SequenceSynthesized primer 3 for mutation of T position in XPA CDR1 869cttgcgctgc ttccggattc bggttctcta agtactttat gttttg 4687046DNAArtificial SequenceSynthesized primer 3 for mutation of T position in XPA CDR1 (antisense) 870gaacgcgacg aaggcctaag vccaagagat tcatgaaata caaaac 4687143DNAArtificial SequenceSynthesized primer 1 for mutation of F4 position in XPA CDR1 871gctgcttccg gattcactnh ttctaagtac tttatgtttt ggg 4387243DNAArtificial SequenceSynthesized primer 1 for mutation of F4 position in XPA CDR1 (antisense) 872cgacgaaggc ctaagtgand aagattcatg aaatacaaaa ccc 4387343DNAArtificial SequenceSynthesized primer 2 for mutation of F4 position in XPA CDR1 873gctgcttccg gattcactva atctaagtac tttatgtttt ggg 4387443DNAArtificial SequenceSynthesized primer 2 for mutation of F4 position in XPA CDR1 (antisense) 874cgacgaaggc ctaagtgabt tagattcatg aaatacaaaa ccc 4387543DNAArtificial SequenceSynthesized primer 3 for mutation of F4 position in XPA CDR1 875gctgcttccg gattcactbg gtctaagtac tttatgtttt ggg 4387643DNAArtificial SequenceSynthesized primer 3 for mutation of F4 position in XPA CDR1 (antisense) 876cgacgaaggc ctaagtgavc cagattcatg aaatacaaaa ccc 4387746DNAArtificial SequenceSynthesized primer 1 for mutation of S position in XPA CDR1 877ctgcttccgg attcactttc nhtaagtact ttatgttttg ggttcg 4687846DNAArtificial SequenceSynthesized primer 1 for mutation of S position in XPA CDR1 (antisense) 878gacgaaggcc taagtgaaag ndattcatga aatacaaaac ccaagc 4687946DNAArtificial SequenceSynthesized primer 2 for mutation of S position in XPA CDR1 879ctgcttccgg attcactttc vaaaagtact ttatgttttg ggttcg 4688046DNAArtificial SequenceSynthesized primer 2 for mutation of S position in XPA CDR1 (antisense) 880gacgaaggcc taagtgaaag bttttcatga aatacaaaac ccaagc 4688146DNAArtificial SequenceSynthesized primer 3 for mutation of S position in XPA CDR1 881ctgcttccgg attcactttc bggaagtact ttatgttttg ggttcg 4688246DNAArtificial SequenceSynthesized primer 3 for mutation of S position in XPA CDR1 (antisense) 882gacgaaggcc taagtgaaag vccttcatga aatacaaaac ccaagc 4688345DNAArtificial SequenceSynthesized primer 1 for mutation of K position in XPA CDR1 883cttccggatt cactttctct nhttacttta tgttttgggt tcgcc 4588445DNAArtificial SequenceSynthesized primer 1 for mutation of K position in XPA CDR1 (antisense) 884gaaggcctaa gtgaaagaga ndaatgaaat acaaaaccca agcgg 4588545DNAArtificial SequenceSynthesized primer 2 for mutation of K position in XPA CDR1 885cttccggatt cactttctct vaatacttta tgttttgggt tcgcc 4588645DNAArtificial SequenceSynthesized primer 2 for mutation of K position in XPA CDR1 (antisense) 886gaaggcctaa gtgaaagaga bttatgaaat acaaaaccca agcgg 4588745DNAArtificial SequenceSynthesized primer 3 for mutation of K position in XPA CDR1 887cttccggatt cactttctct bggtacttta tgttttgggt tcgcc 4588845DNAArtificial SequenceSynthesized primer 3 for mutation of K position in XPA CDR1 (antisense) 888gaaggcctaa gtgaaagaga vccatgaaat acaaaaccca agcgg 4588945DNAArtificial SequenceSynthesized primer 1 for mutation of Y position in XPA CDR1 889ccggattcac tttctctaag nhttttatgt tttgggttcg ccaag 4589045DNAArtificial SequenceSynthesized primer 1 for mutation of Y position in XPA CDR1 (antisense) 890ggcctaagtg aaagagattc ndaaaataca aaacccaagc ggttc 4589145DNAArtificial SequenceSynthesized primer 2 for mutation of Y position in XPA CDR1 891ccggattcac tttctctaag vaatttatgt tttgggttcg ccaag 4589245DNAArtificial SequenceSynthesized primer 2 for mutation of Y position in XPA CDR1 (Artificial Sequence) 892ggcctaagtg aaagagattc bttaaataca aaacccaagc ggttc 4589345DNAArtificial SequenceSynthesized primer 3 for mutation of Y position in XPA CDR1 893ccggattcac tttctctaag bggtttatgt tttgggttcg ccaag 4589445DNAArtificial SequenceSynthesized primer 3 for mutation of Y position in XPA CDR1 (antisense) 894ggcctaagtg aaagagattc vccaaataca aaacccaagc ggttc 4589544DNAArtificial SequenceSynthesized primer 1 for mutation of F8 position in XPA CDR1 895ggattcactt tctctaagta cnhtatgttt tgggttcgcc aagc 4489644DNAArtificial SequenceSynthesized primer 1 for mutation of F8 position in XPA CDR1 (antisense) 896cctaagtgaa agagattcat gndatacaaa acccaagcgg ttcg 4489744DNAArtificial SequenceSynthesized primer 2 for mutation of F8 position in XPA CDR1 897ggattcactt tctctaagta cvaaatgttt tgggttcgcc aagc 4489844DNAArtificial SequenceSynthesized primer 2 for mutation of F8 position in XPA CDR1 (antisense) 898cctaagtgaa agagattcat gbtttacaaa acccaagcgg ttcg 4489944DNAArtificial SequenceSynthesized primer 3 for mutation of F8 position in XPA CDR1 899ggattcactt tctctaagta cbggatgttt tgggttcgcc aagc 4490044DNAArtificial SequenceSynthesized primer 3 for mutation of F8 position in XPA CDR1 (Artificial Sequence) 900cctaagtgaa agagattcat gvcctacaaa acccaagcgg ttcg 4490154DNAArtificial SequenceSynthesized H1-A combination primer 901cttgcgctgc ttccggattc actttctcta aatactttat gttttgggtt cgcc 5490254DNAArtificial SequenceSynthesized H1-B combination primer 902cttgcgctgc ttccggattc actttctctc ygtactttat gttttgggtt cgcc 5490354DNAArtificial SequenceSynthesized H1-C combination primer 903cttgcgctgc ttccggattc actttctcty attactttat gttttgggtt cgcc 5490454DNAArtificial SequenceSynthesized H1-D combination primer 904cttgcgctgc ttccggattc actttctctt ggtactttat gttttgggtt cgcc 5490559DNAArtificial SequenceSynthesized H2-A combination primer 905gtgggtttct gttatctctc ctaaaggtmt catgactcgt tatgctgact ccgttaaag 5990659DNAArtificial SequenceSynthesized H2-B combination primer 906gtgggtttct gttatctctc ctaaaggtma aatgactcgt tatgctgact ccgttaaag 5990759DNAArtificial SequenceSynthesized H2-C

combination primer 907gtgggtttct gttatctctc ctaaaggtsg tatgactcgt tatgctgact ccgttaaag 5990859DNAArtificial SequenceSynthesized H2-D combination primer 908gtgggtttct gttatctctc cttctggtmt catgactcgt tatgctgact ccgttaaag 5990959DNAArtificial SequenceSynthesized H2-E combination primer 909gtgggtttct gttatctctc cttctggtma aatgactcgt tatgctgact ccgttaaag 5991059DNAArtificial SequenceSynthesized H2-F combination primer 910gtgggtttct gttatctctc cttctggtsg tatgactcgt tatgctgact ccgttaaag 5991154DNAArtificial SequenceSynthesized H3-A combination primer 911ctattgtgcg agagtcggcc tgggtgkgaa tycagactac tggggccagg gaac 5491254DNAArtificial SequenceSynthesized H3-B combination primer 912ctattgtgcg agagtcggcc tgggtgkgaa tgaggactac tggggccagg gaac 5491354DNAArtificial SequenceSynthesized H3-C combination primer 913ctattgtgcg agagtcggcc tgggtgkggy gycagactac tggggccagg gaac 5491454DNAArtificial SequenceSynthesized H3-D combination primer 914ctattgtgcg agagtcggcc tgggtgkggy ggaggactac tggggccagg gaac 5491554DNAArtificial SequenceSynthesized H3-E combination primer 915ctattgtgcg agagtcggct atggtgkgaa tycagactac tggggccagg gaac 5491654DNAArtificial SequenceSynthesized H3-F combination primer 916ctattgtgcg agagtcggct atggtgkgaa tgaggactac tggggccagg gaac 5491754DNAArtificial SequenceSynthesized H3-G combination primer 917ctattgtgcg agagtcggct atggtgkggy gycagactac tggggccagg gaac 5491854DNAArtificial SequenceSynthesized H3-H combination primer 918ctattgtgcg agagtcggct atggtgkggy ggaggactac tggggccagg gaac 5491921DNAArtificial SequenceSynthesized H1 fwd combination primer 919gcttccggat tcactttctc t 2192021DNAArtificial SequenceSynthesized H1- rev combination primer 920agagaaagtg aatccggaag c 2192120DNAArtificial SequenceSynthesized H2- fwd combination primer 921gggtttctgt tatctctcct 2092220DNAArtificial SequenceSynthesized H2- rev combination primer 922aggagagata acagaaaccc 2092319DNAArtificial SequenceSynthesized H3- fwd combination primer 923ctattgtgcg agagtcggc 1992420DNAArtificial SequenceSynthesized H3- rev combination primer 924gccgactctc gcacaatagt 2092520DNAArtificial SequenceSynthesized L1R combination primer 925actcgcccga caaatgatgg 2092620DNAArtificial SequenceSynthesized L2R combination primer 926gatcaggagc ttaggggcat 2092721DNAArtificial SequenceSynthesized L3R combination primer 927ctgctgacaa tagtaagttg c 2192858DNAArtificial SequenceSynthesized L127Q28SLW30N combination primer 928ccatcatttg tcgggcgagt cagtbgatta ataggtggtt agcctggtat cagcagac 5892958DNAArtificial SequenceSynthesized L127Q28D30N combination primer 929ccatcatttg tcgggcgagt caggacatta ataggtggtt agcctggtat cagcagac 5893058DNAArtificial SequenceSynthesized L127Q28SLW30F combination primer 930ccatcatttg tcgggcgagt cagtbgattt ttaggtggtt agcctggtat cagcagac 5893158DNAArtificial SequenceSynthesized L127Q28D30F combination primer 931ccatcatttg tcgggcgagt caggacattt ttaggtggtt agcctggtat cagcagac 5893258DNAArtificial SequenceSynthesized L127SF28SLW30N combination primer 932ccatcatttg tcgggcgagt tyttbgatta ataggtggtt agcctggtat cagcagac 5893358DNAArtificial SequenceSynthesized L127SF28D30N combination primer 933ccatcatttg tcgggcgagt tytgacatta ataggtggtt agcctggtat cagcagac 5893458DNAArtificial SequenceSynthesized L127SF28SLW30F combination primer 934ccatcatttg tcgggcgagt tyttbgattt ttaggtggtt agcctggtat cagcagac 5893558DNAArtificial SequenceSynthesized L127SF28D30F combination primer 935ccatcatttg tcgggcgagt tytgacattt ttaggtggtt agcctggtat cagcagac 5893658DNAArtificial SequenceSynthesized L127G28SLW30N combination primer 936ccatcatttg tcgggcgagt ggatbgatta ataggtggtt agcctggtat cagcagac 5893758DNAArtificial SequenceSynthesized L127G28D30N combination primer 937ccatcatttg tcgggcgagt ggagacatta ataggtggtt agcctggtat cagcagac 5893858DNAArtificial SequenceSynthesized L127G28SLW30F combination primer 938ccatcatttg tcgggcgagt ggatbgattt ttaggtggtt agcctggtat cagcagac 5893958DNAArtificial SequenceSynthesized L127G28D30F combination primer 939ccatcatttg tcgggcgagt ggagacattt ttaggtggtt agcctggtat cagcagac 5894055DNAArtificial SequenceSynthesized L251GA53S combination primer 940atgcccctaa gctcctgatc cattctgsta cctctctgca aagtggggtc ccatc 5594155DNAArtificial SequenceSynthesized L251GA53KR combination primer 941atgcccctaa gctcctgatc cattctgsta ccargctgca aagtggggtc ccatc 5594257DNAArtificial SequenceSynthesized L392S93ED combination primer 942gcaacttact attgtcagca ggcttcagak ttcbcatkga ctttcggcgg agggacc 5794357DNAArtificial SequenceSynthesized L392S93S combination primer 943gcaacttact attgtcagca ggcttcatcg ttcbcatkga ctttcggcgg agggacc 5794457DNAArtificial SequenceSynthesized L392D93ED combination primer 944gcaacttact attgtcagca ggctgatgak ttcbcatkga ctttcggcgg agggacc 5794557DNAArtificial SequenceSynthesized L392D93S combination primer 945gcaacttact attgtcagca ggctgattcg ttcbcatkga ctttcggcgg agggacc 57946104PRTArtificial SequenceSynthesized Ca5 light chain variable region 946Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Leu Thr Xaa Ile Cys Arg Ala Ser Gln Asp Ile Asn Arg Trp 20 25 30Leu Ala Trp Gln Gln Thr Pro Gly Asn Ala Pro Lys Leu Leu Ile His 35 40 45Ser Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Met Asp Phe65 70 75 80Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu Thr Phe Gly 85 90 95Gly Gly Thr Lys Val Glu Ile Lys 100947106PRTArtificial SequenceSynthesized A8.2 light chain variable region 947Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ala Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Thr Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Arg Phe Pro Phe Thr 85 90 95Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105948118PRTArtificial SequenceSynthesized Ca5 heavy chain variable region 948Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30Phe Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser Gly Gly Met Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Gly Phe Gly Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115949118PRTArtificial SequenceSynthesized A8.2 heavy chain variable region 949Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Tyr Pro Ser Gly Gly Ile Thr Trp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln Gly Gly Pro Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115950768DNAArtificial SequenceSynthesized ING-1 variable region DNA 950ggcgcgccta accatctatt tcaaggagac agtcataatg aaatacctat tgcctacggc 60agccgctgga ttgttattac tcgctgccca accagcgatg gcgcagatcc agttggtgca 120gtctggacct gagctgaaga agcctggaga gacagtcaag atctcctgca aggcttctgg 180atataccttc acaaaatatg gaatgaactg ggtgaagcag gctccaggaa agggtttaaa 240gtggatgggc tggataaaca cctacactga agagcctaca tatggtgatg acttcaaggg 300acggtttgcc ttctctttgg aaacctctgc cagcactgcc aatttgcaga tcaacaacct 360caaaagtgag gacacggcta catatttctg tgcaagattt ggctctgctg tggactactg 420gggtcaagga acctcggtca ccgtctcctc agcctccacc aagggcccat cggtcttccc 480cctggcaccc tcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa 540ggactacttc cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt 600tcataccttc ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac 660cgtgccctcc agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag 720caacaccaag gtggacaaga gagttgagcc caaatcttgt gcggccgc 768951768DNAArtificial SequenceSynthesized ING-1 variable region DNA (antisense) 951ccgcgcggat tggtagataa agttcctctg tcagtattac tttatggata acggatgccg 60tcggcgacct aacaataatg agcgacgggt tggtcgctac cgcgtctagg tcaaccacgt 120cagacctgga ctcgacttct tcggacctct ctgtcagttc tagaggacgt tccgaagacc 180tatatggaag tgttttatac cttacttgac ccacttcgtc cgaggtcctt tcccaaattt 240cacctacccg acctatttgt ggatgtgact tctcggatgt ataccactac tgaagttccc 300tgccaaacgg aagagaaacc tttggagacg gtcgtgacgg ttaaacgtct agttgttgga 360gttttcactc ctgtgccgat gtataaagac acgttctaaa ccgagacgac acctgatgac 420cccagttcct tggagccagt ggcagaggag tcggaggtgg ttcccgggta gccagaaggg 480ggaccgtggg aggaggttct cgtggagacc cccgtgtcgc cgggacccga cggaccagtt 540cctgatgaag gggcttggcc actgccacag caccttgagt ccgcgggact ggtcgccgca 600agtatggaag ggccgacagg atgtcaggag tcctgagatg agggagtcgt cgcaccactg 660gcacgggagg tcgtcgaacc cgtgggtctg gatgtagacg ttgcacttag tgttcgggtc 720gttgtggttc cacctgttct ctcaactcgg gtttagaaca cgccggcg 768952116PRTArtificial SequenceSynthesized ING-1 KABAT HEAVY CHAIN (KABAT NUMBERING) 952Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Glu Glu Pro Thr Tyr Gly Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Asn65 70 75 80Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Phe Gly Ser Ala Val Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser 115953116PRTArtificial SequenceSynthesized ING-1 CHOTHIA HEAVY CHAIN 953Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Glu Glu Pro Thr Tyr Gly Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Asn65 70 75 80Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Phe Gly Ser Ala Val Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser 115954116PRTArtificial SequenceSynthesized ING-1 IMGT HEAVY CHAIN 954Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Glu Glu Pro Thr Tyr Gly Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Asn65 70 75 80Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Phe Gly Ser Ala Val Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser 115955113PRTArtificial SequenceSynthesized ING-1 KABAT LIGHT CHAIN (KABAT NUMBERING) 955Gln Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly1 5 10 15Thr Ser Gly Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 100 105 110Arg 956113PRTArtificial SequenceSynthesized ING-1 CHOTHIA LIGHT CHAIN 956Gln Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly1 5 10 15Thr Ser Gly Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 100 105 110Arg 957113PRTArtificial SequenceSynthesized ING-1 IMGT LIGHT CHAIN 957Gln Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly1 5 10 15Thr Ser Gly Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 100 105 110Arg 958118PRTArtificial SequenceSynthesized XPA23 KABAT HEAVY CHAIN (KABAT NUMBERING) 958Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30Phe Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser Gly Gly Met Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Gly Tyr Gly Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115959118PRTArtificial SequenceSynthesized XPA23 CHOTHIA HEAVY CHAIN 959Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30Phe Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser Gly Gly Met Thr Arg Tyr Ala Asp Ser Val 50

55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Gly Tyr Gly Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115960118PRTArtificial SequenceSynthesized XPA23 IMGT HEAVY CHAIN 960Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30Phe Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser Gly Gly Met Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Gly Tyr Gly Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115961108PRTArtificial SequenceSynthesized XPA23 KABAT LIGHT CHAIN (KABAT NUMBERING) 961Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Leu Thr Ile Ile Cys Arg Ala Ser Gln Asp Ile Asn Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Thr Pro Gly Asn Ala Pro Lys Leu Leu Ile 35 40 45His Ser Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105962108PRTArtificial SequenceSynthesized XPA23 CHOTHIA LIGHT CHAIN 962Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Leu Thr Ile Ile Cys Arg Ala Ser Gln Asp Ile Asn Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Thr Pro Gly Asn Ala Pro Lys Leu Leu Ile 35 40 45His Ser Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105963108PRTArtificial SequenceSynthesized XPA23 IMGT LIGHT CHAIN 963Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Leu Thr Ile Ile Cys Arg Ala Ser Gln Asp Ile Asn Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Thr Pro Gly Asn Ala Pro Lys Leu Leu Ile 35 40 45His Ser Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 10596464DNAArtificial SequenceSynthesized Representative CDR region 964gtctttcttg cgctgcttcc ggattcactt tctctaagta ctttatgttt tgggttcgcc 60aagc 6496564DNAArtificial SequenceSynthesized Representative CDR region (antisense) 965cagaaagaac gcgacgaagg cctaagtgaa agagattcat gaaatacaaa acccaagcgg 60ttcg 6496657DNAArtificial SequenceSynthesized CDR region 1 966gtctttcttg cgctgcttcc ggattcactt tctctaagta ctttatgttt tgggttc 5796757DNAArtificial SequenceSynthesized CDR region 1 (antisense) 967cagaaagaac gcgacgaagg cctaagtgaa agagattcat gaaatacaaa acccaag 5796853DNAArtificial SequenceSynthesized CDR region 2 968gctgcttccg gattcacttt ctctaagtac tttatgtttt gggttcgcca agc 5396953DNAArtificial SequenceSynthesized CDR region 2 (antisense) 969cgacgaaggc ctaagtgaaa gagattcatg aaatacaaaa cccaagcggt tcg 5397053DNAArtificial SequenceSynthesized R2-5-NHT primer 970gctgcttccg gattcacttt cnhtaagtac tttatgtttt gggttcgcca agc 5397153DNAArtificial SequenceSynthesized R2-5-VAA primer 971gctgcttccg gattcacttt cvaaaagtac tttatgtttt gggttcgcca agc 5397253DNAArtificial SequenceSynthesized R2-5-BGG primer 972gctgcttccg gattcacttt cbggaagtac tttatgtttt gggttcgcca agc 5397353DNAArtificial SequenceSynthesized R2-6-NHT primer 973gctgcttccg gattcacttt ctctnhttac tttatgtttt gggttcgcca agc 5397453DNAArtificial SequenceSynthesized R2-6-VAA primer 974gctgcttccg gattcacttt ctctvaatac tttatgtttt gggttcgcca agc 5397553DNAArtificial SequenceSynthesized R2-6-BGG primer 975gctgcttccg gattcacttt ctctbggtac tttatgtttt gggttcgcca agc 5397653DNAArtificial SequenceSynthesized R2-7-NHT primer 976gctgcttccg gattcacttt ctctaagnht tttatgtttt gggttcgcca agc 5397753DNAArtificial SequenceSynthesized R2-7-VAA primer 977gctgcttccg gattcacttt ctctaagvaa tttatgtttt gggttcgcca agc 5397853DNAArtificial SequenceSynthesized R2-7-BGG primer 978gctgcttccg gattcacttt ctctaagbgg tttatgtttt gggttcgcca agc 5397953DNAArtificial SequenceSynthesized R2-8-NHT primer 979gctgcttccg gattcacttt ctctaagtac nhtatgtttt gggttcgcca agc 5398053DNAArtificial SequenceSynthesized R2-8-VAA primer 980gctgcttccg gattcacttt ctctaagtac vaaatgtttt gggttcgcca agc 5398153DNAArtificial SequenceSynthesized R2-8-BGG primer 981gctgcttccg gattcacttt ctctaagtac bggatgtttt gggttcgcca agc 53

Claims

1. A method for enhancing the binding affinity of a variable domain of an antibody to a binding partner, to obtain a modified variable domain with enhanced binding affinity to the binding partner, the method comprising:a. identifying the proximity assigned to amino acid positions in the variable domain of the antibody using the "prox" line as shown in FIGS. 3A, 3B, 3C or 3D;b. substituting one or more contacting (C), peripheral (P), supporting (S), and/or interfacial (I) amino acid residues with other amino acid residues, thereby generating a library or an array of modified variable domains;c. screening the library or the array for binding affinity to the binding partner; andd. obtaining a modified variable domain with enhanced binding affinity to the binding partner.

2-3. (canceled)

4. A method of making a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising:a. modifying the nucleotide sequence of an antibody variable domain at one or more positions that encode a contacting (C), peripheral (P), supporting (S) and/or interfacial (I) residue identified from the "prox" line as shown in FIGS. 3A, 3B, 3C or 3D to produce amino acid substitutions at the C, P, S and/or I residue thereby generating a library or an array of modified antibody variable domains; andb. selecting a modified variable domain from the library or the array that has enhanced binding affinity to the binding partner compared to the parent variable domain.

5. (canceled)

6. A method for selecting a modified variable domain of an antibody with enhanced binding affinity to a binding partner compared to a parent variable domain, the method comprising:a. obtaining a library or an array of modified antibody variable domains comprising amino acid substitutions at one or more contacting (C), peripheral (P), supporting (S) and/or interfacial (I) residues identified from the "prox" line as shown in FIGS. 3A, 3B, 3C or 3D;b. determining the binding affinity of the modified antibody variable domains and the parent variable domain to the binding partner; andc. selecting the modified antibody variable domains that have enhanced binding affinity to the binding partner compared to the parent variable domain.

7-31. (canceled)

32. A method of mutagenesis of a parent nucleic acid encoding an antibody variable domain to generate modified antibody variable domains, said method comprising:(a.) obtaining one or more primers that each comprise at least one 2 to 12 fold degenerate codon, wherein each primer comprises at least two oligonucleotide sequences that are complementary to a sequence in the parent nucleic acid and code for an amino acid mutation with the exception of cysteine or methionine at one amino acid position encoded by the parent nucleic acid; and(b.) mutating the parent nucleic acid by replication or polymerase based amplification using the one or more primers obtained in (a),wherein replication or amplification of the parent nucleic acid with the one or more primers generates mutated nucleic acids that encode modified antibody variable domains.

33. A method for mutagenesis of an antibody variable domain to obtain modified antibody variable domains with mutated amino acid sequences, the method comprising:a. identifying one or more amino acid positions in the antibody variable domain for mutagenesis;b. substituting one or more of the identified amino acid residues in the antibody variable domain with other amino acid residues excluding cysteine and methionine to generate a library or an array of modified antibody variable domains with mutated amino acid sequences;c. screening the library or array of modified antibody variable domains in an assay for a biological activity of the antibody variable domain; andd. obtaining modified antibody variable domains having the biological activity of the antibody variable domain.

34-36. (canceled)

37. A method of producing a nucleic acid library with an equal representation of non-redundant amino acid changes at an amino acid position encoded by a parent nucleic acid encoding an antibody variable domain, the method comprising:(a.) providing a set of primers that each comprise at least one degenerate codon, wherein each primer comprises at least two oligonucleotide sequence that are complementary to a sequence in the parent nucleic acid and code for an amino acid mutation with the exception of cysteine and methionine at one amino acid position encoded by the parent nucleic acid, wherein the primers code for an equal representation of non-redundant amino acid changes at the one position;(b.) hybridizing a primer from the set to the parent nucleic acid;(c.) replicating or amplifying the parent nucleic acid molecule with the primer to generate nucleic acids that code for amino acid changes at the one position;(d.) repeating steps (b) and (c) with each remaining primer from the set;(e.) pooling the nucleic acids produced with each primer; and(f.) obtaining a library of nucleic acids from steps (a)-(e) coding for an equal representation of amino acid changes at the one position.

38. (canceled)

39. A method of making modified antibody variable domains with mutated amino acid sequences, the method comprising:a. modifying the amino acid sequence of an antibody variable domain to produce amino acid mutations at an amino acid residue in the antibody variable domain to generate a library or an array of modified antibody variable domains with mutated amino acid sequences, wherein the amino acid mutations exclude cysteine and methionine; andb. selecting modified antibody variable domains from the library or the array that have a biological activity of an unmodified antibody variable domain.

40-71. (canceled)

72. A library or an array comprising variants of a antibody variable domain sequence, wherein the variants each comprise an amino acid mutation at one amino acid position in the sequence of a parent antibody variable domain and wherein the amino acid mutations are not cysteine or methionine.

73-76. (canceled)

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