WO2017214211A1

WO2017214211A1 - Methods for identifying a high affinity antibody

Info

Publication number: WO2017214211A1
Application number: PCT/US2017/036235
Authority: WO
Inventors: Daniel Schindler
Original assignee: Igc Bio, Inc.
Priority date: 2016-06-09
Filing date: 2017-06-07
Publication date: 2017-12-14

Abstract

The invention relates to methods for identifying an antibody having a high binding affinity for an antigen. Specifically, the invention relates to determining a frequency of occurrence with respect to binding to an antigen, in order to identify an antibody having a high binding affinity for an antigen.

Description

METHODS FOR IDENTIFYING A HIGH AFFINITY ANTIBODY

FIELD OF THE INVENTION

[0001] The invention relates to methods for identifying an antibody having a high binding affinity for an antigen. Specifically, the invention relates to determining a frequency of occurrence with respect to binding to an antigen, in order to identify an antibody having a high binding affinity for an antigen.

BACKGROUND OF THE INVENTION

[0002] Frequently, in biological investigations, the affinity of a protein for another molecule, such as another protein, a small molecule like a drug, or even a modified amino acid like phosphotyrosine is measured. Current screening methods involve assaying each binding pair individually, which restrict the number of binding pairs to be measured in the hundreds. By binding the pairs and then sequencing by next generation sequencing, the number of pairs which can be analyzed can range from tens of thousands to hundreds of thousands. The effect of the amino acid sequence of the protein on binding can be measured in a binding experiment where all the proteins are all competing for binding to a single molecule. The relative binding affinities can be measured digitally by assessing the relative frequencies of bound molecules by sequencing millions of bound molecules and comparing to the starting mixture.

[0003] For example, a library of antibodies consists of many members, many of which differ by a single amino acid. These antibodies bind to antigens, and the effect of their amino acid sequences on binding an antigen can be measured in a binding experiment where all the antibodies are all competing for binding to a single antigen. The relative binding affinities can be measured digitally by assessing the relative frequencies of bound antibodies by sequencing millions of bound antibodies and comparing to the starting mixture. This method can be applied not only to antibodies, but also to any binding reaction of a protein coded for by a nucleic acid. Examples of other binding reactions are enzymes binding to substrates or transition state analogs, SH2 domains which recognize specific phosphopeptide sequences, or soybean trypsin inhibitor binding to trypsin or other proteases. [0004] The key is to genetically link a defined nucleic acid sequence to a specific protein whose binding is to be tested. In this way a protein's function is physically linked to the gene that encodes it. Linking the phenotype to the genotype in this way takes advantage of the huge advances in DNA sequencing. This linkage can be done with phage, E. coli, emulsions, and yeast. The advantage of yeast is the extensive processing the protein goes through before being finally displayed on the surface. An example of this method was developed by Scott and Smith by binding proteins to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli. See Scott et al., 1990, Science. 1990, vol. 249 (4967), pages 386-90. [0005] However, there exists a need for improved methods for identifying a protein, such as an antibody, having a high binding affinity for a molecule, such as an antigen.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides a method for identifying an antibody for an antigen, the method comprising: providing an antibody library comprising a plurality of antibodies; screening said library against said antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen, thereby identifying said antibody for said antigen.

[0007] In another aspect, the invention provides a kit for identifying an antibody, the kit comprising: an antibody library comprising a plurality of antibodies; instructions for screening said library against an antigen; one or more materials for sequencing a plurality of bound antibodies; a computer implemented media or system for analyzing amino acid sequences to identify one or more amino acid residues associated with a binding affinity for an antibody.

[0008] In yet another aspect, the invention provides a method for enhancing a binding affinity of a predetermined antibody, the method comprising: providing an antibody library comprising a plurality of variant antibodies for said predetermined antibody; screening said library against its antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen.

[0009] In yet another aspect, the invention provides a method for optimizing a binding affinity of a predetermined antibody, the method comprising: providing an antibody library comprising a plurality of variant antibodies for said predetermined antibody; screening said library against its antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen; analyzing said sequences to identify one or more amino acid residues associated with a binding affinity for said antigen; and identifying or selecting a target antibody having said one or more amino acid residues associated with said binding affinity. [0010] In yet another aspect, the invention provides a method for identifying a ligand for a peptide, the method comprising: providing a ligand library comprising a plurality of ligands; screening said library against said peptide; separating a plurality of bound ligands that bound to said peptide from unbound ligands; sequencing said plurality of bound ligands; based on the sequences of said plurality of bound ligands, determining a frequency of occurrence of a ligand within said plurality of bound antibodies, wherein the most frequently occurring ligand represents a ligand having the highest affinity to said peptide, thereby identifying said ligand for said peptide.

[0011] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The invention provides methods for identifying an antibody having a high binding affinity for an antigen. Specifically, the invention relates to determining a frequency of occurrence with respect to binding to an antigen, in order to identify an antibody having a high binding affinity for an antigen.

[0013] One of skilled in the art will appreciate that any type of antibody expression library (e.g., VH or VL domain expression library) can be used in the methods of the invention. Examples of suitable expression libraries include, but not limited to, nucleic acid display, phage display, retroviral display, and cell surface display libraries (e.g., yeast, mammalian, and bacterial cells). In a particular embodiment, the library is a yeast display library.

[0014] Methods for screening expression libraries are well known in the art and fully described in U.S. Patent Application Publications US2014/0113831 and US 2010/0168393, which are incorporated by reference herein in their entirety.

[0015] Libraries according to the invention can be used for direct screening using the genetic and/or target ligands or used in a selection protocol that involves a genetic display package. Yeast or bacteriophage lambda expression systems may be screened directly using the techniques well known in the art. [0016] Other suitable screening systems can also be used. In some embodiments, the screening systems may rely, for example, on direct chemical synthesis of library members. In one example, the method involves the synthesis of peptides on a set of pins or rods, as described in WO84/03564, which is incorporated by reference herein in its entirety. In another example, the method involves peptide synthesis on beads, which forms a peptide library in which each bead is an individual library member, as described in US Patent 4,631,211 and PCT Patent Application Publications WO92/00091 and WO93/06121, which are incorporated by reference herein in their entirety.

[0017] In yet another example, the screening system may use the synthesis of arrays of peptides on a surface in a manner that places each distinct library member (e.g., unique amino acid sequence) at a discrete, predefined location in the array. The locations in the array where binding interactions between a predetermined molecule (e.g., antigen) and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location. These methods are described, for example, in US Patent Application Publication US 20100168393 ; US Patent 5,143,854; PCT Patent Application Publications WO90/15070 and WO92/10092; and scientific publications Fodor et al. (1991) Science, 251 : 767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26: 271, which are incorporated by reference herein in their entirety.

[0018] In another embodiment, a selection display system can be used for the construction of libraries of the invention, as described, for example, in US Patent Application Publication US 20100168393. Any suitable selection display system, known to one of skilled in the art, may be used in conjunction with a library according to the invention. Selection protocols for isolating desired members of large libraries are well known in the art, as exemplified by phage display techniques. One advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Additionally, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.

[0019] Sequencing methods are well known in the art. Any suitable sequencing method can be used. Numerous sequence analysis tools are available and well known in the art.

[0020] In one embodiment, the invention provides analyzing amino acid sequences. In another embodiment, the invention provides analyzing nucleic acid sequences. In a particular embodiment, the invention provides analyzing amino acid sequences to identify one or more amino acid residues associated with a binding affinity to, for example, an antigen. In a particular embodiment, the invention provides identifying or selecting a target antibody having the one or more amino acid residues associated with the binding affinity.

[0021] The antibody library of the invention may include recombinant vectors. The vector may comprise a nucleic acid encoding only one antibody chain or a portion thereof (e.g. , the heavy or light chain) or a nucleic acid encoding both antibody chains or portions thereof. [0022] Vector can be any suitable vector known to one of skilled in the art. In a particular embodiment, the vector is a plasmid vector. The term "plasmid," as used herein may refer to a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within a suitable host. [0023] In one example, the plasmid is a yeast plasmid. Yeast is organism that naturally harbour plasmids. Both circular and linear plasmids are encompassed within the scope of the invention. In one embodiment, the plasmid is a Yeast integrative plasmid (Yip). In some embodiments, Yip is a vector that relies on integration into the host chromosome for survival and replication. Yip may also be associated with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C).

[0024] In another embodiment, the plasmid is a Yeast Replicative Plasmid (YRp). In some embodiments, YRp transports a sequence of chromosomal DNA that includes an origin of replication. [0025] Other suitable vectors are also within the scope of the invention. Other exemplary vectors include, but not limited to, phagemids, cosmids, viruses and phage nucleic acids or other nucleic acid molecules that are capable of replication in a prokaryotic or eukaryotic host.

[0026] The vectors typically contain a marker to provide a phenotypic trait for selection of transformed hosts. In one example, any suitable selectable marker, known to one of skilled in the art can be used. The term "selectable marker," as used herein, may refer to a gene introduced into a cell that confers a trait suitable for artificial selection. In one example, the selectable marker is a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign nucleic acid into a cell.

[0027] In one embodiment, the selectable marker is a positive selectable marker. Positive selection marker may confer selective advantage to the host organism. In one example, the positive selectable marker is an antibiotic resistant gene, which allows the host organism to survive antibiotic selection. The host cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those colonies that can grow have successfully taken up and expressed the introduced genetic material. In one embodiment, the positive selectable marker may confer resistance to antibiotics such as, for example, ampicillin, neomycin, chloramphenicol, tetracycline, or kanamycin.

[0028] In another embodiment, the selectable marker is a negative selectable marker. Negative or counterselectable markers are selectable markers that eliminate or inhibit growth of the host organism upon selection. An example of a negative selectable marker includes thymidine kinase, which makes the host sensitive to ganciclovir selection.

[0029] In yet another embodiment, the selectable marker is a combination of positive and negative selectable marker. Such marker can serve as both a positive and a negative marker by conferring an advantage to the host under one condition, but inhibits growth under a different condition. In one example, the combination of positive and negative selectable marker includes an enzyme that can complement an auxotrophy (positive selection) and be able to convert a chemical to a toxic compound (negative selection).

[0030] Additional examples of selectable markers include, but not limited to, beta-lactamase which confers ampicillin resistance to bacterial hosts; neo gene from Tn5, which confers resistance to kanamycin and G418; and a gene coding for a mutant Fabl protein (mfabl), which confers triclosan resistance to the host.

[0031] In a particular embodiment, the selectable marker is a yeast marker, for example, URA3, an orotidine-5' phosphate decarboxylase from yeast, which is a positive and negative selectable marker.

[0032] Useful markers for other expression systems, are well known to those of skilled in the art. These and other selectable markers can be obtained from commercially available plasmids, using techniques well known in the art.

[0033] The nucleic acid sequence of a selectable marker may be of any suitable length. In a particular embodiment, the length of said selectable marker nucleic acid sequence ranges from approximately 370 bp (zeo,bleoR) to approximately 860 bp (amp).

[0034] In one example, the start codon is a standard AUG (or ATG) codon, found in both prokaryotes and eukaryotes. In another example, the start codon is a non-AUG (or non- ATG) codon. Alternate start codons (non AUG) are very rare in eukaryotic genomes. However, naturally occurring non-AUG start codons have been reported for some cellular mRNAs. See Ivanov et ah, 2011, Nucleic Acids Research vol. 39 (10), pages 4220^234.

[0035] The selectable marker or any gene of interest in the invention may also comprise a stop codon. In the genetic code, a stop codon (or termination codon) may refer to a nucleotide triplet within messenger RNA that signals a termination of translation. Examples of stop codon include, for example, but not limited to UAG, UAA, and UGA.

[0036] In one embodiment, the vector may be an expression vector, wherein the nucleic acid encoding the antibody is operably linked to an expression control sequence. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid molecules of the invention. The vectors may also contain genetic expression cassettes containing an independent terminator sequence, sequences permitting replication of the vector in both eukaryotes and prokaryotes, i.e. , shuttle vectors and selection markers for both prokaryotic and eukaryotic systems. When the vector may contain nucleic acids encoding both a heavy and light chain or portions thereof, the nucleic acid encoding the heavy chain may be under the same or a separate promoter. The separate promoters may be identical or may be different types of promoters.

[0037] Suitable promoters include constitutive promoters and inducible promoters. Representative expression control sequences/promoters include, for example, the glycolytic promoters of yeast, e.g. , the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha mating factors, the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, promoters derived from the human cytomegalovirus, metallothionine promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters of SV40.

[0038] In some embodiments, promoters useful in yeast expression systems include, for example, promoters from sequences encoding enzymes in the metabolic pathway such as alcohol dehydrogenase (ADH) (EPO Publication No. 284,044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO Publication No. 329,203) promoters. In some embodiments, the expression construct comprises a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734), as well as promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EPO Publication No. 164,556). These and other promoters can be obtained from commercially available plasmids, using techniques well known in the art.

[0039] In another embodiment, transcription termination and polyadenylation sequences are also present in the expression constructs. These sequences are located 3' to the translation stop codon for the coding sequence. Transcription terminator/polyadenylation signal sequences are well known in the art.

[0040] A host of the present invention may be eukaryotic or prokaryotic. Suitable eukaryotic cells include yeast and other fungi, insect cells, plant cells, human cells, and animal cells, including mammalian cells, such as hybridoma lines, COS cells, NS0 cells and CHO cells. Suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.

[0041] The terms "host cell", as used herein, may refer to a cell or population of cells into which a nucleic acid molecule or vector of the invention is introduced. "A population of host cells" refers to a group of cultured cells into which a nucleic acid molecule or vector of the present invention can be introduced and expressed. The host may contain a nucleic acid or vector encoding only one chain or portion thereof (e.g. , the heavy or light chain); or it may contain a nucleic acid or vector encoding both chains or portions thereof, either an the same or separate nucleic acids and/or vectors.

[0042] Nucleic acid molecules comprising nucleotide sequences of interest can be stably integrated into a host cell genome or maintained on a stable episomal element in a suitable host cell using various gene delivery techniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346. A number of appropriate host cells for use with the above systems are also known. For example, yeast hosts useful in the present invention include, but not limited to, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, and Spodoptera frugiperda. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney cells, human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others.

[0043] In another aspect, the present invention can be used in expression constructs to express a wide variety of substances. In an exemplary embodiment, the present invention is used to express an antibody or an antigen-binding fragment thereof. In one embodiment, the present invention is used to express humanized recombinant antibody. In one embodiment, the invention is used to express the nucleic acid sequence that encodes a heavy or light chain immunoglobulin.

[0044] The vector of the invention can be constructed by methods or techniques well known in the art. Construction of yeast strains are well known and fully disclosed, for example, in U.S. Patent 5,635,369 and U.S. Patent Application Publication 2002/0160380, which are incorporated by reference herein in their entirety. Plasmid construction by homologous recombination yeast is also known in the art. See e.g. , Ma et al, 1987, Gene, vol. 58, pages 201-216, which is incorporated by reference herein in its entirety.

[0045] A wide variety of methods, known to one of skilled in the art, can be used to deliver the expression constructs to cells. Such methods include, for example, but are not limited to, DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyornithine-mediated transfection, electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinjection.

[0046] In another aspect, provided herein is a method for optimizing or enhancing a binding affinity of a predetermined antibody, the method comprising: providing an antibody library comprising a plurality of variant antibodies for said predetermined antibody; screening said library against its antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen. [0047] Any known or commercially available antibody can be optimized for its binding affinity. Examples of an antibody that can be optimized by the invention include, but are not limited to anti-EGFR antibodies such as Erbitux (cetuximab), ABX-EGF, and Merck Mab 425; IGF-1R antibodies, for example, cixutumumab, figitumumab, dalotuzumab, ganitumab, R1507, SCH717454, AVE1642, BIIB022, and MEDI-573 ; anti-17-lA cell surface antigen antibodies such as Panorex (edrecolomab); anti-4-lBB antibodies; anti-IL4 DC antibodies; anti-A33 antibodies such as A33 and CDP-833; anti-.alpha.4.beta.l integrin antibodies such as natalizumab; anti-complement factor 5 (C5) antibodies such as 5G1.1 ; anti-CA125 antibodies such as OVAREX (oregovomab); anti-CD3 antibodies such as NUVION (visilizumab) and Rexomab; anti-CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B and Oncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodies such as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7, 2H7.vl6, 2H7.vl l4, 2H7.vl l5, BEXXAR (tositumomab, 1-131 labeled anti- CD20), RITUXAN (rituximab), and ZEVALIN (Ibritumomab tiuxetan, Y-90 labeled anti- CD20); anti-CD22 antibodies such as LYMPHOCIDE (epratuzumab, Y-90 labeled anti- CD22); anti-CD23 antibodies such as IDEC-152; anti-CD25 antibodies such as basiliximab and ZENAPAX (daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30; anti-CD33 antibodies such as MYLOTARG (gemtuzumab ozogamicin), Oncolysin M, and Smart M195 ; anti-CD38 antibodies; anti-CD40 antibodies such as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8, ANTOVA, and IDEC-131; anti-CD44 antibodies such as bivatuzumab; anti-CD46 antibodies; anti-CD52 antibodies (alemtuzumab); anti-CD55 antibodies such as SC-1 ; anti-CD56 antibodies such as huN901-DMl ; anti-CD64 antibodies such as MDX-33; anti-CD66e antibodies such as XR-303 ; anti-CD74 antibodies such as IMMU-110; anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89 antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodies such as B-B4-DM1 ; anti- CD146 antibodies such as AA-98; anti-CD148 antibodies; anti-CEA antibodies such as CT84.66, labetuzumab, and PENTACEA; anti-CTLA-4 antibodies such as MDX-101 ; anti- CXCR4 antibodies; anti-EpCAM antibodies such as Crucell's anti-EpCAM, ING-1, and IS- IL-2; anti-ephrin B2/EphB4 antibodies; anti-Her2 antibodies such as HERCEPTIN, MDX- 210; anti-FAP (fibroblast activation protein) antibodies such as sibrotuzumab; anti-ferritin antibodies such as NXT-211 ; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250 antibodies such as WX- G250 and RENCAREX; anti-GD2 ganglioside antibodies such as EMD-273063 and TriGem; anti-GD3 ganglioside antibodies such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIa antibodies such as ReoPro; anti-heparinase antibodies; anti-HLA antibodies such as ONCOLYM, Smart 1D10; anti-HM1.24 antibodies; anti-ICAM antibodies such as ICM3; anti-IgA receptor antibodies; anti-IGF-1 antibodies such as CP-751871 and EM- 164; anti- IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such as CNTO-328 and elsilimomab; anti-IL-15 antibodies such as HUMAX-IL15; anti-KDR antibodies; anti- laminin 5 antibodies; anti- Lewis Y antigen antibodies such as Hu3S193 and IGN-311 ; anti- MCAM antibodies; anti-Mucl antibodies such as BravaRex and TriAb; anti- NC AM antibodies such as ERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn and Therex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptk antibodies; anti- PTN antibodies; anti-RANKL antibodies such as AMG-162; anti-RLIP76 antibodies; anti- SK-1 antigen antibodies such as Monopharm C; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA and MDX-220; anti-TGF-.beta. antibodies such as CAT- 152; anti-TNF-.alpha. antibodies such as CDP571, CDP870, D2E7, HUMIRA (adalimumab), and REMICADE (infliximab); anti-TRAIL-Rl and TRAIL-R2 antibodies; anti-VE-cadherin-2 antibodies; anti-VLA-4 antibodies; antibodies to treat autoimmune or inflammatory disease; antibodies to treat transplant rejection; antibodies to treat infectious diseases, for example, anti-anthrax antibodies such as ABthrax, anti-CMV antibodies such as CytoGam and sevirumab, anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G, anti- helicobacter antibodies such as Pyloran, anti-hepatitis B antibodies such as HepeX-B, Nabi- HB, anti-HIV antibodies such as HRG-214, anti-RSV antibodies such as felvizumab, HNK- 20, palivizumab, RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb, BSYX-A110, and SE-Mab or their combinations.

[0048] The invention also includes methods of producing an antibody of the present invention, which entails culturing a host cell expressing one or more nucleic acid sequences encoding an antibody of the present invention, and recovering the antibody from the culture medium. In certain embodiments, the antibody is purified by separating it from the culture medium. Antibodies comprising more than one chain can be produced by expressing each chain together in the same host; or as separate chains, which are assembled before or after recovery from the culture medium. [0049] The invention also provides a kit for identifying an antibody. The kit may comprise an antibody library comprising a plurality of antibodies; instructions for screening said library against an antigen; one or more materials for sequencing a plurality of bound antibodies; a computer implemented media or system for analyzing amino acid sequences to identify one or more amino acid residues associated with a binding affinity for an antibody.

[0050] The term "nucleic acid," as used herein, can include both double- and single-stranded sequences and refers to, but not limited to, cDNA from yeast, viral, procaryotic or eucaryotic mRNA, genomic DNA sequences, or procaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.

[0051] The term "recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0052] The term "host cell," as used herein, refers to a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.

[0053] The term "expression construct," as used herein, refer to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. The expression construct includes control elements, as described above, such as a promoter or promoter/enhancer which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression construct described herein may be contained within a plasmid construct. In addition to the components of the expression construct, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single- stranded DNA (e.g., an origin of replication).

[0054] As used herein, the term "antibody" includes intact immunoglobulin molecules comprising 4 polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CHI , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa () and lambda (λ), based on the amino acid sequences of their constant domains. The variable regions of kappa light chains are referred to herein as VK. The expression of VL, as used herein, is intended to include both the variable regions from kappa-type light chains (VK) and from lambda-type light chains. The light chain constant region is comprised of one domain, CL. The VH and VL regions include regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. "CDRH1" refers to the first CDR region in an antibody heavy chain, "CDRH2" refers to the second CDR region in an antibody heavy chain, and "CDRH3" refers to the third CDR region in an antibody heavy chain. "CDRL1" refers to the first CDR region in an antibody light chain, "CDRL2" refers to the second CDR region in an antibody light chain, and "CDRL3" refers to the third CDR region in an antibody light chain.

[0055] The term "antibody" as used herein is also intended to encompass intact antibodies, functional fragments which bind antigen, and variants thereof which bind antigen, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof; each containing at least one CDR. Antibodies of the invention include antibody fragments or variants having one, two, three, four, five, six or more CDR regions.

[0056] Antibody fragments which are embraced by the present invention include Fab (e.g. , by papain digestion), Facb (e.g. , by plasmin digestion), pFc' (e.g. , by pepsin or plasmin digestion), Fd (e.g. , by pepsin digestion, partial reduction and reaggregation), sVd, and Fv or scFv (e.g. , by molecular biology techniques). Antibody fragments are also intended to include domain deleted antibodies, diabodies, triabodies, linear antibodies, single-chain antibody molecules (including camelized antibodies), and multispecific antibodies formed from antibody fragments. The term "linear antibodies," as used herein, refers to the antibodies described in Zapata et al , Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (p_j -Cyl- Vp_j -Cyl) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0057] The term "antibody," as used herein, also includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g. , mouse or rat) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Thus, the present invention includes, for example, chimeric antibodies comprising a chimeric heavy chain and/or a chimeric light chain. The chimeric heavy chain may comprise any of the heavy chain variable (VH) regions described herein or mutants or variants thereof fused to a heavy chain constant region of a non-human antibody. The chimeric light chain may comprise any of the light chain variable (VL) regions described herein or mutants or variants thereof fused to a light chain constant region of a non-human antibody.

[0058] Antibodies of the invention also include "humanized antibodies", which are antibody molecules having one or more complementarity determining regions (CDRs) from a non- human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, or improve, antigen binding. These framework substitutions are identified standard techniques such as by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. Antibodies can be humanized using a variety of techniques including CDR-grafting, veneering or resurfacing, and chain shuffling.

[0059] The term "human antibody," as used herein, includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The human antibody can have at least one position replaced with an amino acid residue, e.g. , an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0060] Antibodies of the invention also include "recombinant human antibody," which includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.

[0061] Antibodies of the present invention can be monospecific, bispecific or multispecific. Monospecific antibodies bind to only one antigen. Bispecific antibodies (BsAbs) are antibodies that have two different antigen-binding specificities or sites. Multispecific antibodies have more than two different antigen-binding specificities or sites. Where an antibody has more than one specificity, the recognized epitopes can be associated with a single antigen or with more than one antigen.

[0062] All patents, patent applications, and scientific publications cited herein are hereby incorporated by reference in their entirety.

[0063] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES EXAMPLE 1

High Multiplexed Digital Competitive Binding Assay

[0064] We have developed a high multiplexed digital competitive binding assay.

[0065] The amino acid sequences of peptides displayed on a phage can be determined by sequencing the corresponding coding region in the viral DNA's. To greatly increase the power of the existing technique two enabling technologies are used. They are the ability to synthesize a large number of specific nucleotide sequences and then to sequence them by next generation sequencing, for example as developed by Illumina or IonTorrent. The nucleotide sequences can even be tagged by short unique sequences to enable a large number to be sequences at one time. In addition sequencing this tag can identify the entire sequence. For example, because of the degeneracy of the genetic code a 6 amino acid stretch (18 nucleotides) consisting of 2 of the amino acids represented by six codons (leu,ser,arg) and 4 amino acids coded for by 4 codons (val, ala, thr, gly, pro) can code for 9,216 different nucleotide sequences [0066] Using a carefully designed starting library of antibody genes and expressing them on the surface of yeast, antibodies were selected against defined protein antigens. The frequency of binders is normalized to the frequency in the original expression library.

[0067] An example of using next-generation sequencing on a phage display library of short peptides is given by "identification of target-binding peptide motifs by high-throughput sequencing of phage-selected peptides" and "deep sequencing analysis of phage libraries using Illumina platform."

[0068] Our method has the following uses: (1) a library of antibodies displayed on yeast or phage, preferably sorted by FACS or magnetic beads or other immobilizing surface on a specific antigen such as a method called panning. The key step is getting a method to separate bound from unbound molecules. When the bound antibodies are sequenced, the tightest binders' sequences will be most frequently occurring. (2) This method can be used to determine the sequence of the antibody and antigen at the same time. A library of antibodies displayed on yeast or phage, preferably sorted by FACS or magnetic beads on a collection of antigens. The antigens are also displayed as a library on yeast or phage. The sequences of the bound antibodies and antigens can be distinguished by sequencing. When the bound antibodies are sequenced, the tightest binders' sequences will be most frequently occurring. The same is true of the antigens displayed on yeast or phage. The most frequently occurring antibody sequence can be compared to the most frequently occurring antigen sequence. Alternatively, the sequence of the most frequently occurring antibody can be used as a probe to select which antibody by checking antigen clones individually and separately. (3) A library of antibodies as above can be selected after a certain amount of time to select by off rate. A capture process based on the off-rate is more powerful as a result of the exponential nature of the curves for dissociation of complexed antibody.

[0069] There are 20 million yeast cells in 1 ml at an OD of 1. Yeast expressing scFv are incubated with biotinylated antigen which will bind to the scFv. Then the cells can be captured on an immobilized surface containing avidin or a derivative biotin binding agent. In fact, using the orthogonal binding agents of anti-biotin antibody and streptavidin in sequential enrichment binding steps to prevent non-specific binding. Generally biotinylated antigens can be purchased premade. However if necessary protein can be biotinylated with a commercial kit. Cells can be labeled at a concentration of 10⁹ cells/ml EXAMPLE 2

[0070] A library of 10⁵ scFv antibodies was screened against antigen A and the binders were selected by FACS. The binders were sequenced by MiSeq sequencing using Nextera. A cluster of closely related antibodies was identified and their frequency of occurrence counted. Antibody 1 and 2 have similar frequencies indicating that an interchange of isoleucine and valine in the light chain does not matter. Replacing 2 serines with a glycine and an aspartate reduces the binding affinity of Ab4 to a third of the standards, ABl and Ab2. Ab3 which replaces S48 with Ala and S56 with Gly almost complete knocks out activity. Although Ab5 can be found in the original library used for binding (the expression library) the single change of a tyrosine in heavy CDR3 to phenylalanine, simply removing the hydroxyl moiety, prevents it from being found in the bound fraction. The first numbers on the left are positions in the heavy chain with little letters representing positions in the complementarity- determining region and the next group of numbers and letters representing positions in the light chain.

48 54 56 i 19 46 h

Abl S S S Y V I R 126

Ab2 S S S Y I V R 123

Ab3 A S G Y V I R 2

Ab4 S G D Y V I R 38

Ab5 S S S F V I R 0

EXAMPLE 3

[0071] As above, but analysis of another cluster. In this cluster amino acid 52 of the heavy chain was proline or serine and position 59 was tyrosine (Y) or histidine (H). In the heavy CDR3 (H-CDR3) amino acid e was tyrosine or histidine (H)and position f was tyrosine or phenylalanine. In the light chain position 29 was arginine (R), glycine (G), asparagine (N), or serine (S). In the light chain CDR3 position c was phenylalanine (F) or tyrosine and position f is phenylalanine of valine. It is noteworthy of all the choices of amino acids at position 29 of the light chain, glycine is strongly represented.

52 59 ef 29 cf

Abl P Y YF G FF 627

Ab2 S H YF G FF 208

Ab3 P Y YF N FF 74

Ab4 S H YY G YF 139

Ab5 P Y YF G YF 135

Ab6 P Y YF G YV 116

Ab7 S H YF S FF 13

Ab8 P Y HY S FF 2

Ab9 S H YY R FF 1

EXAMPLE 4

[0072] As above, but in this experiment even though the sequencing results are from the same sequencing run it is a selection on a different library. This demonstrates the large effect of changing histidine at position d of the heavy CDR3 to a phenylalanine (F). Even though Ab9 is well represented in the original expression library it is totally absent in the selected antibodies even though it differs in only 2 positions from Abl, a serine (S) instead of a histidine in position 34 of the heavy chain and a serine instead of a tyrosine (Y) in position 32 of the light chain.

34 78 de 32

Abl H L HY Y 246780

Ab2 H L FY Y 2

Ab3 S Ά HY Y 1615

Ab4 S V HY Y 6

Ab5 H L YH Y 31

Ab6 H L YF Y 17

Ab7 S L HY Y 5

Ab8 H L YH S 1

Ab9 S L HY S 0

EXAMPLE 5

[0073] In this example an IGHV3_23 heavy chain framework was identified with a histidine replacing the natural serine at position 34 or the natural serine. At position 78 an alanine or valine can replace the natural leucine. In the HCDR3 a phenylalanine can replace the natural tyrosine at position 5 , a histidine can replace the tyrosine at position 6, an alanine can replace the serine at position 8 and a serine can replace the threonine at position 9. In the light chain a tyrosine can replace a lysine at position 29 and a tyrosine can replace the serine at position 32. Abl binds the epitope strongly while replacing the aromatic, partially hydrophobic tyrosine at light chain position 32 with a disfavored substitution serine greatly reduces binding.

[0074] Histidine is unique with its chemical properties and can only substitute partially with tyrosine as indicated by a BLOSUM matrix (BLOcks Substitution Matrix). Replacing the histidine at heavy chain position 34 with a serine greatly reduces binding. Putting both of these changes together in the same molecules reduces binding even more to 324 units. Any alteration in the H-CDR3 greatly reduces binding.

34 78 efhi 29 32

Abl H L YYAT K Y 120695

Ab2 H L YYAT K S 8657

Ab3 S L YYAT K Y 4545

Ab4 S L YYAT K S 324

Ab5 H L YYSS K Y 611

Ab6 H L FYST K Y 160

Ab7 H L YHST K Y 80

Ab8 H L YYAT Y Y 51

Ab9 H L YYST K Y 27

AblO H L YYAT K s 26

Abll S Ά YYSS K s 15

Abl2 H L YFST K Y 4

Abl3 S Ά YYAT Y s 3

Abl4 S Ά YHST K Y 2

Abl5 S V FYST K Y 1

Abl 6 S V YYAT K Y 1

Abl7 S Ά FYST K Y 1 EXAMPLE 6

[0075] In this example, an IGHV3_23 heavy chain framework was identified with either an asparagine or aspartate replacing the natural serine at position 30 or an alanine replacing the natural leucine at position 78. The different HCDR3s used were varied at positions 4, 7 and 12 and where the leucine at position 4 was replaced by isoleucine or valine, the serine at position 7 was replaced by asparagine or alanine, or the valine at position 12 was replaced by tyrosine. Replacing the tyrosine in position 12 with valine totally abrogated binding. Replacing the serine at position 30 with asparagine had little effect on binding but removing the amide group and introducing a negative charge with aspartate greatly reduced binding as evidenced by Ab4. Replacing the hydrophobic leucine at position 78 with the small alanine with only a methyl side chain almost completely destroyed binding. Any single change in the HCDR3 abrogated binding.

30 78 dgl

Abl S L LSY 75103

Ab2 S L LSV 3

Ab3 N L LSY 69649

Ab4 D L LSY 5213

Ab5 S Ά LSY 382

Ab6 S L ISV 44

Ab7 S L L V 43

Ab8 N L L V 9

Ab9 N L ISV 7

AblO N L VSV 6

Abll S L VSV 5

Abl2 S L LSV 3

Abl3 S Ά LNV 3

Abl4 D L LNV 1

Abl5 D L ISV 1

Abl 6 S L LAV 1

[0076] Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying an antibody for an antigen, the method comprising: providing an antibody library comprising a plurality of antibodies; screening said library against said antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen, thereby identifying said antibody for said antigen.

2. The method of claim 1, wherein the sequences of said plurality of bound antibodies are amino acid sequences.

3. The method of claim 2, further comprising the steps of analyzing said amino acid sequences and identifying one or more amino acid residues associated with a binding affinity for said antigen.

4. The method of claim 3, further comprising the step of identifying or selecting a target antibody having said one or more amino acid residues associated with said binding affinity.

5. The method of claim 1 , wherein said antibody library comprises a plurality of vector.

6. The method of claim 5, wherein said vector is a plasmid vector.

7. The method of claim 6, wherein said plasmid is a self-replicating plasmid.

8. The method claim 5, wherein said vector is a yeast vector.

9. The method claim 1, wherein the step screening is performed by a yeast display system.

10. The method claim 5, wherein said vector is a bacteriophage vector.

11. The method claim 1, wherein the step screening is performed by a phage display system.

12. The method claim 1 , wherein phage display is a bacteriophage display.

13. The method claim 12, wherein said phage is an affinity phage that displays tight- binding of a plurality of antibodies.

14. The method of claim 5, each vector comprises the nucleic acid sequence encoding the amino acid sequence of at least one of said plurality of antibodies.

15. The method of claim 1, wherein the step of separating comprises separating host cells expressing said bound antibodies from host cells expressing said unbound antibodies.

16. The method of claim 1 , wherein said antibody library comprises one or more single- chain variable fragments (scFv).

17. The method of claim 1 , wherein said antibody library comprises one or more antibody fragments.

18. The method of claim 17, wherein at least one of said fragments comprises an amino acid sequence of complementarity determining region (CDR).

19. The method of claim 18, wherein said CDR is a CDR of heavy chain H3 (CDRH3).

20. The method of claim 1, wherein said bound antibodies are sequenced by a high- throughput sequencing.

21. The method of claim 1 , wherein said frequency of occurrence is determined based a predetermined threshold level.

22. The method of claim 1, wherein said binding affinity is a predetermined binding affinity.

23. The method of claim 1, wherein said one or more amino acid residues associated with said binding affinity are determined based on digitally aligning the amino acid sequences of said bound antibodies and associating each aligned sequence with said frequency of occurrence.

24. An antibody identified by a method of claim 1.

25. A kit for identifying an antibody, the kit comprising: an antibody library comprising a plurality of antibodies; instructions for screening said library against an antigen; one or more materials for sequencing a plurality of bound antibodies; a computer implemented media or system for analyzing amino acid sequences to identify one or more amino acid residues associated with a binding affinity for an antibody.

26. A method for enhancing a binding affinity of a predetermined antibody, the method comprising: providing an antibody library comprising a plurality of variant antibodies for said predetermined antibody; screening said library against its antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen.

27. A method for optimizing a binding affinity of a predetermined antibody, the method comprising: providing an antibody library comprising a plurality of variant antibodies for said predetermined antibody; screening said library against its antigen; separating a plurality of bound antibodies that bound to said antigen from unbound antibodies; sequencing said plurality of bound antibodies; based on the sequences of said plurality of bound antibodies, determining a frequency of occurrence of an antibody within said plurality of bound antibodies, wherein the most frequently occurring antibody represents an antibody having the highest affinity to said antigen; analyzing said sequences to identify one or more amino acid residues associated with a binding affinity for said antigen; and identifying or selecting a target antibody having said one or more amino acid residues associated with said binding affinity.

28. A method for identifying a ligand for a peptide, the method comprising: providing a ligand library comprising a plurality of ligands; screening said library against said peptide; separating a plurality of bound ligands that bound to said peptide from unbound ligands; sequencing said plurality of bound ligands; based on the sequences of said plurality of bound ligands, determining a frequency of occurrence of a ligand within said plurality of bound antibodies, wherein the most frequently occurring ligand represents a ligand having the highest affinity to said peptide, thereby identifying said ligand for said peptide.