Peptide-MHC compacts

Abstract

To provide various compositions and processes for producing peptide-MHC multimers.SOLUTION: According to an embodiment, a polynucleotide molecule is provided, comprising, in a 5' to 3' orientation, (i) a first universal target sequence, (ii) a nucleotide sequence encoding an antigenic peptide, (iii) a second universal target sequence that is distinct from the first universal target sequence, (iv) a Beta 2 Microglobulin sequence, and (v) a Multiple Histocompatability Complex (MHC) allele sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 651,639, filed April 2, 2018, which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created in XX / 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] T cells are the primary mediators of adaptive immunity. Guided by the specificity of each T cell's unique T cell receptor (TCR), T cells regulate autoimmunity, assist in the activation of B cells and innate effectors, and directly kill infected and cancerous cells in a precisely targeted manner. Each TCR recognizes ligands presented by major histocompatibility complex (MHC) molecules on target cells. Identifying relevant peptide-MHC complex ligands plays a role in understanding immune responses to tumors and pathogens. MHC complex ligands are also useful for understanding responses to self-antigens and dietary antigens. This understanding enables clinically beneficial immunotherapies (e.g., TCR gene transfer and vaccines) that initiate, amplify, or attenuate immune responses to target antigens.

[0004] Mutated "neoepitopes" are important targets for endogenous and engineered immune responses against cancer. Neoepitope-reactive TILs are present in the endogenous repertoire and induce tumor regression upon adoptive transfer. Similarly, tumor mutational burden predicts the clinical efficacy of CTLA-4 or PD-1 blockade, suggesting that these checkpoint inhibition strategies affect tumor regression by liberating neoepitope-reactive T cells. Because neoepitopes result from somatic mutations in tumor cells, they are not commonly presented by thymic epithelial cells to induce central immune tolerance. Therefore, T cell responses directed at these neoepitopes are tumor-specific, likely high-affinity, and patient-specific (i.e., private). From a clinical perspective, this presents both an opportunity and a challenge: neoepitopes are excellent targets for immunotherapy, but TCR isolation methods should be sufficiently high-throughput to enable therapeutic application on a clinically useful scale.

[0005] There is an unmet need for rapid and robust TCR ligand discovery technologies for both basic and translational research. Peptide-MHC multimers enable the sorting of T cells according to the antigen specificity of their TCRs, a key step in isolating tumor-specific TCRs for gene therapy. A typical current peptide-MHC generation protocol begins with solid-phase synthesis of the desired peptide ligand(s). In parallel, universal β2-microglobulin and related MHC class I molecules are heterologously expressed in Escherichia coli, resulting in misfolded inclusion bodies. Each peptide is added to a refolding reaction containing β2M and related MHC class I molecules. Finally, the portion of the ternary complex that refolds correctly can be purified and formulated for use in peptide-MHC multimer generation. Schumacher and colleagues devised a photocleavable peptide that binds to a specific MHC molecule as a conditional ligand to facilitate the parallel generation of specific MHC molecules with many different peptide ligands. A single refolding reaction generates MHC molecules bound to the conditional ligand. When exposed to UV light, the conditional ligand is cleaved and exchanged with the desired peptide, which is present in excess. Many such exchange reactions can be performed in parallel, enabling the construction of pMHC libraries for that specific MHC allele. Nevertheless, this state-of-the-art technology has significant limitations. First, the generation, purification, and refolding of MHC molecules expressed in E. coli inclusion bodies is challenging, and properly folded peptide-MHC complexes are produced in low yield. Second, the turnaround time (weeks) for commercial peptide synthesis is not optimal for the context of personalized, on-demand TCR gene therapy directed at patient-specific neoepitopes. Third, many predicted ligands cannot be used to screen T cells through this approach because the biophysical properties of the peptide (e.g., hydrophobicity) prevent their synthesis or exchange. Fourth, exchange efficiencies are generally low (<50% for the majority of predicted HLA-binding peptides).A mixture of properly folded, exchanged MHC and misfolded, unliganded MHC results in multimer staining with low signal-to-noise. This problem is exacerbated when screening T cells with multiplexed peptide-MHC reagent pools. Fifth, designing and validating ligands for each new MHC allele is a difficult and non-robust task. Because the MHC locus is the most multiply allelic locus in the human genome, this poses a significant obstacle to neoepitope-targeted gene therapy across patients with diverse MHC haplotypes. Collectively, these limitations highlight the need for novel technologies in this field. Disclosed herein are various compositions and methods for generating peptide-MHC multimers that address these limitations. Summary of the Invention

[0006] The methods and compositions described herein enable rapid identification of antigens targeted by an immune response and isolation of T cells, and thus the TCR sequences mediating that response. This rapid identification is a goal of research and drug development in the fields of infectious disease, tumor immunology, autoimmunity, and immunotherapy. The compositions can be applied to identify and isolate antigen-specific T cells and their encoded MHC I-restricted TCRs for drug development. Identification and characterization of antigen-specific T cells can also be applied to the diagnosis, monitoring, and prognosis of immune responses and disease in treatment settings (pre- and post-treatment). The comPACT approach can also be applied to identify targets of MHC II-restricted T cell responses. Such responses are central to autoimmune diseases and are emerging as a key component of cancer immunotherapy. Furthermore, the efficiency of comPACT protein secretion from mammalian cell expression data generated by the methods described herein is potentially useful as a surrogate metric of proper protein folding and, therefore, the affinity of each neoepitope ligand for its cognate MHC molecule. This technology, in turn, may offer the unique ability to refine MHC binding algorithms for less well-studied MHC alleles and improve other targeted immunotherapies, such as neoepitope vaccines. Thus, the compositions disclosed herein have broad application in human health and clear commercial potential.

[0007] In one aspect, a polynucleotide molecule disclosed herein comprises, in the 5' to 3' direction, (i) a first universal target sequence, (ii) a nucleotide sequence encoding an antigenic peptide, (iii) a second universal target sequence different from the first universal target sequence, (iv) a beta 2 microglobulin (β2M) sequence, and (v) a multiple histocompatibility complex (MHC) allele sequence. In one embodiment, the polynucleotide molecule comprises, from 5' to 3', (i) a promoter sequence, (ii) a first universal target sequence comprising the sequence set forth in SEQ ID NO: 3, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target sequence comprising the sequence set forth in SEQ ID NO: 4, (v) a β2M sequence comprising the sequence set forth in SEQ ID NO: 106, (vi) an MHC allele sequence comprising a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 109-174, (vii) a first affinity tag sequence comprising the sequence set forth in SEQ ID NO: 29, (vii) a protease cleavage site sequence comprising the sequence set forth in SEQ ID NO: 31, and (ix) a second affinity tag sequence comprising the sequence set forth in SEQ ID NO: 35.

[0008] In one embodiment, the antigenic peptide is selected from the group consisting of a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phosphoantigen, and a microbial antigen. In another embodiment, the antigenic peptide is a neoantigen.

[0009] In some embodiments, the neoantigen is selected by analyzing tumor sequencing data from the subject to identify one or more somatic mutations. In one embodiment, the analysis is performed using an in silico prediction algorithm. In another embodiment, the prediction algorithm further comprises an MHC binding algorithm for predicting binding between the neoantigen and an MHC allele.

[0010] In some embodiments, the MHC allele is a mammalian MHC allele. In some embodiments, the MHC allele is a human MHC allele. In some embodiments, the MHC allele is a class I HLA allele. In other embodiments, the HLA allele comprises an HLA-A, HLA-B, or HLA-C allele. The HLA allele is an HLA allele of a subject.

[0011] In some embodiments, the HLA alleles are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A* 29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, H LA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40: 02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA- B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*0 7:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01.

[0012] In some embodiments, the HLA allele comprises a sequence selected from the group consisting of SEQ ID NOs: 109-174.

[0013] In some embodiments, the β2M allele is a mammalian β2M allele. In some embodiments, the β2M allele is a human β2M allele. In some embodiments, the β2M allele comprises the sequence set forth in SEQ ID NO:106.

[0014] In some embodiments, the nucleotide sequence encoding the antigenic peptide is 20 to 60, 20 to 30, 25 to 35, 20 to 45, 30 to 45, 40 to 60, or 45 to 60 nucleotides in length. In one embodiment, the nucleotide sequence encoding the antigenic peptide is 20 to 30 nucleotides in length.

[0015] In some embodiments, the first universal target sequence is 4 to 50, 4 to 15, 15 to 40, 15 to 35, 15 to 30, 20 to 40, 25 to 40, or 30 to 40 nucleotides in length. In one embodiment, the first universal target sequence is 25 to 35 nucleotides in length. In another embodiment, the first universal target sequence is at least about 15 nucleotides in length. In another embodiment, the first universal target sequence is 4 to 6 nucleotides in length. In some embodiments, the second universal target sequence is 4 to 50, 4 to 15, 15 to 40, 15 to 35, 15 to 30, 20 to 40, 25 to 40, or 30 to 40 nucleotides in length. In one embodiment, the second universal target sequence is 25 to 35 nucleotides in length. In another embodiment, the second universal target sequence is at least about 15 nucleotides in length. In another embodiment, the second universal target sequence is 4 to 6 nucleotides in length.

[0016] In some embodiments, the first and second universal target sequences comprise polymerase chain reaction (PCR) primer target sequences, while in other embodiments, the first and second universal target sequences comprise restriction enzyme cleavage sites.

[0017] In some embodiments, the sequence of the first universal target comprises the sequence set forth in SEQ ID NO: 3. In some embodiments, the sequence of the second universal target comprises the sequence set forth in SEQ ID NO: 4.

[0018] In some embodiments, the first universal target sequence further comprises a signal sequence. In some embodiments, the signal sequence encodes a signal sequence including a human growth hormone signal sequence, a hIG1 kappa light chain signal sequence, a beta2 microglobulin signal sequence, or an IL2 signal sequence. In some embodiments, the signal sequence comprises a sequence comprising the sequence set forth in SEQ ID NO: 1, 24, 26, or 28. In one embodiment, the signal sequence encodes human growth hormone (HGH). In some embodiments, the signal sequence comprises the sequence set forth in SEQ ID NO: 1. In one embodiment, the signal sequence is 40 to 90, 40 to 60, 45 to 70, 50 to 80, 60 to 90, 55 to 70, 60 to 80, or 70 to 80 nucleotides in length.

[0019] In some embodiments, the 3' end of the polynucleotide sequence further comprises, in a 5' to 3' direction, a purification cluster sequence comprising: (i) a first affinity tag sequence, (ii) a protease cleavage site sequence, and (iii) a second affinity tag sequence. In some embodiments, the first and second affinity tags are selected from the group consisting of AviTag, streptavidin-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and Myc-tag. In some embodiments, the first affinity tag comprises a sequence comprising the sequence set forth in SEQ ID NO:29, and the second affinity tag comprises a sequence comprising the sequence set forth in SEQ ID NO:33.

[0020] In some embodiments, the protease cleavage site sequence is selected from the group consisting of a TEV cleavage site sequence, a thrombin cleavage site sequence, a factor Xa cleavage site sequence, an enteropeptidase cleavage site sequence, and a rhinovirus 3C protease cleavage site sequence. In one embodiment, the protease cleavage site sequence comprises the TEV cleavage site sequence set forth in SEQ ID NO: 31. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a TEV cleavage site, and the second affinity tag encodes a His6 peptide. In one embodiment, the first affinity tag sequence comprises the sequence set forth in SEQ ID NO: 29, the protease cleavage site comprises the sequence set forth in SEQ ID NO: 31, and the second affinity tag comprises the sequence set forth in SEQ ID NO: 33.

[0021] In some embodiments, the second universal target sequence further comprises a first linker sequence. In one embodiment, the first linker sequence comprises the sequence set forth in SEQ ID NO: 10, 14, 16, or 18.

[0022] In some embodiments, the polynucleotide further comprises a second linker sequence between the β2M sequence and the MHC allele sequence. In one embodiment, the second linker sequence comprises the sequence set forth in SEQ ID NO: 10 or 20.

[0023] In some embodiments, the polynucleotide further comprises a third linker sequence between the MHC allele sequence and the first affinity tag. In one embodiment, the third linker sequence comprises the sequence set forth in SEQ ID NO: 12 or 22.

[0024] The 5' end of the polynucleotide sequence may further comprise a promoter sequence linked to the 5' end of the first universal target. In some embodiments, the promoter sequence is a CMV, EF1α, or SV40 promoter. In one embodiment, the promoter sequence is a CMV promoter. In one embodiment, the promoter sequence is a CMV promoter sequence.

[0025] The 3' end of the polynucleotide sequence can further comprise a polyA sequence. In some embodiments, the polyA sequence is an SV40, hGH, BHG, or rbGlob polyA sequence. In one embodiment, the polyA sequence comprises the bGH polyA sequence set forth in SEQ ID NO: 179.

[0026] In one embodiment, the polynucleotide molecule comprises, from 5' to 3', (i) a promoter sequence, (ii) a first universal target sequence, (iii) a nucleotide sequence encoding an antigenic peptide, (iv) a second universal target sequence different from the first universal target sequence, (v) a β2M sequence, (v) an MHC allele sequence, (vi) a first affinity tag sequence, (vii) a protease cleavage site sequence, and (vii) a second affinity tag sequence.

[0027] In another aspect, described herein is an expression construct comprising a polynucleotide molecule disclosed herein. In one embodiment, the expression construct comprises a plasmid or viral vector.

[0028] In another aspect, described herein is a host cell comprising a polynucleotide molecule or expression construct, as described above. In one embodiment, the polynucleotide is integrated into the cell genome. In another embodiment, the polynucleotide is extrachromosomal. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In one embodiment, the cell is a stem cell, a tumor cell, an immortalized cell, or a fetal cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell expresses a BirA protein or a fragment thereof.

[0029] In yet another aspect, the present specification describes a library comprising the polynucleotide molecules or expression constructs described herein, wherein the library comprises two or more different polynucleotide molecules, each different polynucleotide molecule comprising (i) a first universal sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is non-identical in each of the two or more polynucleotide molecules, (iii) a second universal target sequence, (iv) a β2M sequence, and (v) an MHC allele sequence. In one embodiment, the MHC allele sequence is non-identical in each of the two or more polynucleotide molecules. In another embodiment, the library comprises 20 to 500 different polynucleotide molecules. In some embodiments, the library comprises at least 66 different polynucleotide molecules.

[0030] In some embodiments, the library comprises at least HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA -A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:0 2, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B* 40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, H LA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03 :04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01 alleles. In some embodiments, the library comprises at least the sequences set forth in SEQ ID NOs: 109-174.

[0031] In another aspect, described herein is a polypeptide comprising, from amino terminus to carboxyl terminus, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide. In some embodiments, the antigenic peptide is selected from the group consisting of a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phosphoantigen, and a microbial antigen. In one embodiment, the antigenic peptide is a neoantigen.

[0032] In some embodiments, the polypeptide comprises, from 5' to 3', (i) a promoter peptide comprising the sequence set forth in SEQ ID NO: 2, (ii) a first universal target peptide, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target peptide comprising the sequence set forth in SEQ ID NO: 15 or 17, (v) a β2M peptide comprising the sequence set forth in SEQ ID NO: 105, (vi) an MHC peptide comprising the sequence set forth in SEQ ID NOs: 38-103, (vii) a first affinity peptide comprising the sequence set forth in SEQ ID NO: 30, (viii) a protease cleavage site peptide comprising the sequence set forth in SEQ ID NO: 32, and (ix) a second affinity peptide comprising the sequence set forth in SEQ ID NO: 36.

[0033] In some embodiments, the neoantigen is selected by analyzing tumor sequencing data from the subject to identify one or more somatic mutations. In one embodiment, the analysis is performed using an in silico prediction algorithm. In another embodiment, the prediction algorithm further comprises an MHC binding algorithm for predicting binding between the neoantigen and an MHC peptide.

[0034] In one embodiment, the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0035] In some embodiments, the MHC peptide is a mammalian MHC peptide. In some embodiments, the MHC peptide is a human MHC peptide. In some embodiments, the MHC peptide is a class I HLA peptide. In other embodiments, the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide. In one embodiment, the HLA peptide is an HLA peptide of interest. In one embodiment, the HLA peptide comprises a Y84A or Y84C mutation.

[0036] In some embodiments, the β2M peptide is a mammalian β2M peptide. In some embodiments, the β2M peptide is a human β2M peptide. In some embodiments, the β2M peptide comprises the sequence set forth in SEQ ID NO: 105. In another embodiment, the β2M peptide comprises an S88C mutation. In some embodiments, the β2M peptide comprises the sequence set forth in SEQ ID NO: 107, which comprises an S88C mutation at amino acid 88.

[0037] In some embodiments, the HLA peptide comprises HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA-C*17:01. In some embodiments, the HLA peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 38 to 103.

[0038] In some embodiments, the first universal target peptide further comprises a signal peptide. In one embodiment, the signal sequence is 15 to 45, 15 to 30, 20 to 45, 20 to 30, or 30 to 45 amino acids in length. In one embodiment, the signal peptide comprises human growth hormone. In some embodiments, the signal peptide comprises human growth hormone signal peptide, hIG1 kappa light chain signal peptide, beta2 microglobulin signal peptide, or IL2 signal peptide. In some embodiments, the signal peptide comprises a sequence comprising the sequence set forth in SEQ ID NO: 2, 23, 25, or 27. In some embodiments, the signal peptide comprises the human growth hormone (HGH) signal peptide sequence set forth in SEQ ID NO: 2. In one embodiment, the second universal target peptide comprises the sequence GGGGSGGGSGGGGGS. In one embodiment, the second universal target peptide comprises the sequence set forth in SEQ ID NO: 15 or 17.

[0039] In some embodiments, the carboxyl terminus of the polypeptide may further comprise a purification cluster comprising, from the amino terminus to the carboxyl terminus, (i) a first affinity peptide, (ii) a protease cleavage site, and (iii) a second affinity peptide. In some embodiments, the first and second affinity peptides are selected from the group consisting of AviTag, Strep-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and / or Myc-tag. In some embodiments, the first affinity peptide comprises a sequence comprising SEQ ID NO: 30, and the second affinity peptide comprises a sequence comprising SEQ ID NO: 34.

[0040] In some embodiments, the protease cleavage site is a TEV cleavage site, a thrombin cleavage site, a factor Xa cleavage site, an enteropeptidase cleavage site, or a rhinovirus 3C protease cleavage site. In some embodiments, the purification cluster comprises an AviTag epitope, a TEV cleavage site, and a His6 epitope. In some embodiments, the protease cleavage site comprises the TEV cleavage site sequence set forth in SEQ ID NO: 32. In some embodiments, the purification cluster comprises the AviTag peptide sequence set forth in SEQ ID NO: 30, the TEV cleavage site sequence set forth in SEQ ID NO: 32, and the His6 peptide sequence set forth in SEQ ID NO: 34 or 36. In some embodiments, the purification cluster comprises two or more copies of the His6 peptide sequence set forth in SEQ ID NO: 34. In some embodiments, the purification cluster comprises the sequence set forth in SEQ ID NO: 37.

[0041] In some embodiments, the second universal target peptide further comprises a linker comprising the sequence set forth in SEQ ID NO: 9, 11, 15, or 17.

[0042] In some embodiments, the polypeptide comprises a second linker sequence between the β2M sequence and the MHC allele sequence. In some embodiments, the second linker sequence comprises the sequence set forth in SEQ ID NO: 9 or 19. In some embodiments, the polypeptide further comprises a third linker sequence between the MHC allele sequence and the first affinity tag. In some embodiments, the third linker sequence comprises the sequence set forth in SEQ ID NO: 13 or 21. In some embodiments, the polypeptide is biotinylated.

[0043] In one embodiment, the polypeptide comprises, from amino terminus to carboxyl terminus, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide, (iv) a β2M peptide, (v) an MHC peptide, (vi) a first affinity tag peptide, (vii) a protease cleavage site, and (vii) a second affinity tag peptide. In some embodiments, the polypeptide is biotinylated.

[0044] In another aspect, disclosed herein is a library comprising two or more different polypeptide molecules, the different polypeptide molecules comprising (i) a first universal target peptide, (ii) an antigenic peptide, where the antigenic peptide is not identical for each of the two or more polypeptide molecules, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide. In one embodiment, the MHC peptide is not identical for each of the two or more polypeptide molecules. In another embodiment, the library comprises 20 to 500 different polypeptide molecules. In some embodiments, the library comprises at least 66 different polypeptides.

[0045] In some embodiments, the library includes HLA-A*01:01、HLA-A*02:01、HLA-A*03:01、HLA-A*24:02、HLA-A*30:02、HLA-A*31:01、HLA-A*32:01、 HLA-A*33:01、HLA-A*68:01、HLA-A*11:01、HLA-A*23:01、HLA-A*30:01、HLA-A*33:03、HLA-A*25:01、HLA-A*26:01、HLA-A*2 9:02、HLA-A*68:02、HLA-B*07:02、HLA-B*14:02、HLA-B*18:01、HLA-B*27:02、HLA-B*39:01、HLA-B*40:01、HLA-B*44:02、HL A-B*46:01、HLA-B*50:01、HLA-B*57:01、HLA-B*58:01、HLA-B*08:01、HLA-B*15:01、HLA-B*15:03、HLA-B*35:01、HLA-B*40: 02、HLA-B*42:01、HLA-B*44:03、HLA-B*51:01、HLA-B*53:01、HLA-B*13:02、HLA-B*15:07、HLA-B*27:05、HLA-B*35:03、HLA -B*37:01、HLA-B*38:01、HLA-B*41:02、HLA-B*44:05、HLA-B*49:01、HLA-B*52:01、HLA-B*55:01、HLA-C*02:02、HLA-C*03:0 4、HLA-C*05:01、HLA-C*07:01、HLA-C*01:02、HLA-C*04:01、HLA-C*06:02、HLA-C*07:02、HLA-C*16:01、HLA-C*03:03、HLA-C *07:04、HLA-C*08:01、HLA-C*08:02、HLA-C*12:02、HLA-C*12:03、HLA-C*14:02、HLA-C*15:02、and HLA-C*17:01 polypeptides.

[0046] In some embodiments, the polypeptide is bound to a particle, and the particle is a surface, a nanoparticle, a bead, or a polymer. In one embodiment, the polypeptide is bound to the particle via a linker. In one embodiment, the particle is a nanoparticle, and the nanoparticle is a magnetic nanoparticle or a polystyrene nanoparticle. In one embodiment, the magnetic nanoparticle comprises magnetic iron oxide. In another embodiment, the bead is an agarose bead or a sepharose bead. In some embodiments, the polypeptide bound to the particle further comprises a fluorophore. In one embodiment, the fluorophore is bound to the particle with or without a linker.

[0047] In some embodiments, a library of polypeptides is bound to particles, the library comprising two or more different polypeptide molecules, the antigen peptide is not identical for each of the two or more polypeptide molecules, and each different polypeptide is bound to a particle. In one embodiment, the MHC peptide is not identical for each of the two or more polypeptide molecules. In one embodiment, the library further comprises a unique, defined barcode sequence operably associated with the identity of each different polypeptide. In another embodiment, the library comprises between 20 and 500 different polypeptides. In another embodiment, the library comprises at least 66 different polypeptides.

[0048] In another aspect, the kits described herein are kits that include the composition of any of the above embodiments and instructions for use.

[0049] In another aspect, disclosed herein is a method for making a polynucleotide molecule, comprising: (a) obtaining a first polynucleotide sequence comprising, in a 5' to 3' direction, (i) a first universal target sequence comprising a restriction enzyme recognition site, (ii) a second universal target sequence comprising a restriction enzyme recognition site that is not identical to the first target sequence, (iii) a β2M sequence, and (iv) an MHC allele sequence; (b) obtaining a second polynucleotide comprising, in a 5' to 3' direction, (i) a portion of the first universal target sequence, (ii) a sequence encoding an antigenic peptide, and (iii) a portion of the second universal target sequence; (c) obtaining a second polynucleotide comprising, in a 5' to 3' direction, (i) a portion of the first universal target sequence, (ii) a sequence encoding an antigenic peptide, and (iii) a portion of the second universal target sequence; The method includes (i) obtaining a third polynucleotide comprising (i) a reverse complement of a portion of the first universal target sequence, (ii) a reverse complement of a sequence encoding an antigenic peptide, and (iii) a reverse complement of a portion of the first universal target sequence; (d) mixing the second and third polynucleotides together so that the complementary sequences anneal; (e) performing restriction digestion of the first polynucleotide sequence with at least one restriction enzyme; and (f) ligating the first and second polynucleotides together by mixing the digested first polynucleotide and the annealed second and third polynucleotides together with DNA ligation reaction reagents.

[0050] In some embodiments, the method further comprises phosphorylating the 5' nucleotide of the second and third polynucleotides after complementary sequence annealing, wherein the phosphorylation comprises incubating the first or the second and third polynucleotides with an enzyme. In some embodiments, the enzyme is T4 kinase. In some embodiments, the first polynucleotide is an expression construct. In some embodiments, the method comprises inserting the produced polynucleotide into an expression construct.

[0051] In some embodiments, the first polynucleotide further comprises a promoter sequence. In one embodiment, the promoter sequence is a CMV, EF1α, or SV40 promoter.

[0052] In another embodiment, the 3' end of the second polynucleotide sequence further comprises a purification cluster sequence comprising, in a 5' to 3' direction, (i) a first affinity tag sequence, (ii) a protease cleavage site sequence, and (iii) a second affinity tag sequence.

[0053] In one embodiment, the 3' end of the second polynucleotide sequence further comprises a polyA sequence.

[0054] In some embodiments, obtaining the third and fourth polynucleotide sequences further comprises obtaining a predicted dataset of tumor sequencing data from the subject. In one embodiment, obtaining the third and fourth polynucleotide sequences further comprises predicting a sequence encoding the antigenic peptide from the tumor sequencing data. In another embodiment, obtaining the third and fourth polynucleotide sequences further comprises synthesizing a polynucleotide encoding the antigenic peptide based on the predicted sequence.

[0055] In some embodiments, the method further includes producing a library comprising two or more different polynucleotide molecules, the different polynucleotide molecules comprising: (i) a first universal target sequence; (ii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide has a nucleotide sequence that is non-identical to each of the two or more polypeptide molecules; (iii) a second universal target sequence that is different from the first universal target sequence; (iv) a β2M peptide; and (v) an MHC allele sequence. In one embodiment, the MHC allele is non-identical to each of the two or more polynucleotide molecules. In another embodiment, the library comprises between 40 and 500 different polynucleotide molecules.

[0056] In some embodiments, the method further comprises inserting the produced polynucleotide into an expression construct. In one embodiment, the method further comprises producing a library comprising two or more expression constructs comprising different polynucleotide molecules, the different polynucleotide molecules comprising (i) a first universal target sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is non-identical in each of the two or more polynucleotide molecules, (iii) a second universal target sequence different from the first universal target sequence, (iv) a β2M sequence, and (v) an MHC allele sequence. In one embodiment, the MHC allele is non-identical in each of the two or more polynucleotide molecules. In another embodiment, the library comprises 20 to 500 different expression constructs. In some embodiments, the library comprises at least 66 different polynucleotide molecules. In some embodiments, the MHC allele is selected from the sequence set forth in the group consisting of SEQ ID NOs: 109-174.

[0057] In some embodiments, the method further comprises expressing the polyprotein molecule from the polynucleotide molecule. In some embodiments, the polynucleotide is transfected or transduced into the cell. In one embodiment, the polynucleotide is integrated into the cell genome. In another embodiment, the polynucleotide remains extrachromosomal in the cell. In some embodiments, the host cell is a mammalian cell or a human cell. In one embodiment, the cell is a stem cell, a tumor cell, an immortalized cell, or a fetal cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is an E. coli cell.

[0058] In one embodiment, the method further comprises quantifying polyprotein expression. In some embodiments, the expressed and non-expressed polyprotein sequences are used to refine the antigenic peptide prediction analysis step of the method. In one embodiment, the method further comprises biotinylating the polyprotein.

[0059] In some embodiments, the method includes purifying the polyprotein. The purification step can include affinity chromatography. In one embodiment, the affinity chromatography includes immobilized metal affinity chromatography, which includes a support, a chelating agent, and a divalent metal. The chelating agent for affinity chromatography can be nitrotriacetic acid (NTA) or iminidiacetic acid (IDA). In some embodiments, the divalent metal is selected from the group consisting of nickel (Ni), cobalt (Co), copper (Cu), and iron (Fe). In one embodiment, the chelating agent is NTA and the divalent metal is Ni. In other embodiments, the support is selected from the group consisting of agarose beads, sepharose beads, and magnetic beads. In some embodiments, the method further includes quantifying the biotinylation level of the purified polyprotein.

[0060] In some embodiments, the method further comprises binding the polypeptide to a particle, wherein the particle is a surface, a nanoparticle, a bead, or a polymer. In one embodiment, the polypeptide is bound to the particle via a linker. In one embodiment, the particle is a nanoparticle, wherein the nanoparticle is a magnetic nanoparticle or a polystyrene nanoparticle. In one embodiment, the magnetic nanoparticle comprises magnetic iron oxide. In another embodiment, the bead is an agarose bead or a sepharose bead. In some embodiments, the polypeptide bound to the particle further comprises a fluorophore. In one embodiment, the fluorophore is bound to the particle with or without a linker.

[0061] In another embodiment, the method further includes generating a library comprising two or more different polyproteins bound to at least one particle, wherein the different polypeptides comprise (i) a first universal target peptide, (ii) an antigenic peptide, wherein the antigenic peptide is not identical for each of the two or more polypeptide molecules, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide. In one embodiment, the MHC peptide is not identical for each of the two or more polypeptide molecules. In another embodiment, the library comprises 20 to 500 different single polyproteins. In another embodiment, the library comprises at least 66 different single polyproteins. In another embodiment, the MHC peptide is selected from the group consisting of SEQ ID NOs: 39-104.

[0062] In another aspect, disclosed herein is a method for producing a polynucleotide molecule, the method comprising: (a) obtaining a first polynucleotide sequence comprising, in a 5' to 3' direction, (i) a first universal target sequence comprising a restriction enzyme recognition site, (ii) a second universal target sequence comprising a restriction enzyme recognition site that is not identical to the first target sequence, (iii) a β2M sequence, and (iv) an MHC allele sequence; (b) obtaining a second polynucleotide sequence comprising, in a 5' to 3' direction, (i) the first universal target sequence, (ii) a sequence encoding an antigenic peptide, and (iii) the second universal target sequence; (c) performing restriction digestion of the first polynucleotide sequence with at least one restriction enzyme; (d) performing restriction digestion of the second polynucleotide sequence with at least one restriction enzyme; and (e) ligating the first and second polynucleotides together by mixing the digested first and second polynucleotides together with DNA ligation reaction reagents.

[0063] In another aspect, described herein is a method for making a polynucleotide molecule, the method comprising: (a) obtaining a first polynucleotide sequence comprising a first universal target sequence; (b) obtaining a second polynucleotide sequence comprising, in the 5' to 3' direction, (i) a second universal target sequence, (ii) a β2M sequence, and (iii) an MHC allele sequence; (c) obtaining a third polynucleotide sequence comprising, in the 5' to 3' direction, a sequence encoding an antigenic peptide and the second universal target sequence; and (d) obtaining a third polynucleotide sequence comprising, in the 5' to 3' direction, a reverse complement of the antigenic peptide and the first universal target sequence. (e) obtaining a fourth polynucleotide sequence comprising the reverse complement of the sequence; (f) combining the first, second, third, and fourth polynucleotides in solution; (g) adding polymerase chain reaction (PCR) reagents; and (g) performing a PCR reaction, wherein complementary regions of the second universal target sequence in the second and third polynucleotides anneal to each other and complementary regions of the first universal target sequence in the first and fourth polynucleotides anneal to each other; the annealed sequences provide priming sequences for the PCR extension and amplification reaction.

[0064] Also disclosed herein is a method for isolating antigen-specific T cells, comprising: (a) providing a polypeptide comprising, from amino terminus to carboxyl terminus, (i) a first universal target peptide, (ii) an antigen peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide, wherein the polypeptide is linked to a particle; (b) providing a sample known or suspected to contain one or more T cells; (c) contacting the polypeptide with the sample, comprising providing conditions sufficient for a single T cell to bind to the polypeptide bound to the particle; and (d) isolating the single T cell bound to the particle.

[0065] These and other features, aspects, and advantages of the disclosed compositions and methods will become better understood with regard to the following description and accompanying drawings. [Brief explanation of the drawings]

[0066] [Figure 1]

[0023] Figure 1 shows an exemplary comPACT minigene design. SS refers to an optional signal sequence; US1 refers to the first universal target site; neoantigen refers to the antigen peptide sequence site; US2 refers to the second universal target site; L1 refers to an optional first linker sequence; β2M refers to the β-2-microglobulin domain sequence; L2 refers to an optional second linker sequence; MHC heavy chain refers to an MHC heavy chain allele; L3 refers to an optional third linker sequence; and purification cluster refers to an optional purification cluster having a biotinylation sequence, a protease cleavage site, and an affinity tag sequence. [Figure 2] FIG. 1 shows a schematic representation of an exemplary modular, off-the-shelf platform for rapidly assembling libraries of antigenic peptide ligands complexed with selected MHC alleles. [Figure 3]

[0033] Figure 1 is a schematic diagram of an exemplary restriction digestion cloning reaction for replacing a dummy insert in an MHC template with a selected neoepitope sequence. The dummy insert (underlined, bold) contains four stop codons in a different frame and unique restriction enzyme recognition sites for destroying the uncut or religated template. Restriction enzyme recognition sites on either side of the insert are shown in boxes. [Figure 4]Figure 1 shows a schematic diagram of an exemplary restriction digestion cloning reaction for inserting a selected neoepitope sequence into an MHC template. The neoepitope sequence (underlined, bold) is synthesized as a primer flanked by two distinct restriction enzyme recognition sites (boxed). A universal primer with the reverse complement of the 3' restriction enzyme recognition site is used in a PCR reaction to form a double-stranded primer-dimer of the neoepitope sequence. Restriction digestion of both the neoepitope and the MHC template vector allows for a ligation reaction to insert the selected neoepitope sequence into the MHC template sequence. The ligation reaction is transformed into E. coli, and the plasmid prepared from the transformed E. coli is used in a mammalian production cell transfection reaction. [Figure 5] Figure 1 shows a schematic diagram of an exemplary alternative restriction digestion cloning reaction for inserting a selected neoepitope sequence into an MHC template. Two complementary NeoE-encoding primers are synthesized with partial 5' and 3' restriction enzyme recognition sites. These primers are annealed to simulate overhangs from restriction digestion. A pre-cut vector (which precisely retains the 5' phosphate group at the overhanging end) is then ligated with the annealed NeoE insert, and the ligation product is transformed into E. coli for plasmid generation. [Figure 6]This diagram illustrates an exemplary PCR-based method for inserting a selected neoepitope sequence into an MHC template. Two complementary NeoE-encoding primers are synthesized: a forward primer with the 3' sequence for the second universal site in the MHC template; and a reverse primer with the 3' sequence for the complementary sequence of the first universal site in the MHC template. These primers are mixed with the 5' fragment of the MHC template containing the first universal site and a second fragment of the MHC template containing the second universal site and the remainder of the comPACT minigene. The first PCR amplification cycle generates two nucleotide fragments: one fragment encodes the first universal site region with the downstream neoepitope, and the other encodes the neoepitope followed by the remainder of the comPACT gene. These two fragments overlap at the unique neoepitope sequence and are then assembled. The complete assembly is amplified and cleaned up for transfection. [Figure 7] Figure 1 shows total protein expression in 30 mL of mammalian cells transfected with the comPACT gene (Neo12) over a 7-day time course and Western blot using NTA-HRP reagent to detect the His-tag. [Figure 8] 1 shows a gel of Ni-NTA affinity chromatography purification of Neo12 comPACT protein, where Pre represents crude lysate, FT represents flow-through, W represents wash, and E represents elution. [Figure 9] 1 shows the size-exclusion chromatography spectrum of purified Neo12 protein, where the major peak is Neo12 protein and the minor peak is ATP added in the biotinylation step. [Figure 10] FIG. 9 shows a purification experiment similar to that shown in FIG. 8, using 0.7 cell culture volumes. [Figure 11] FIG. 1 shows crude and purified proteins of eight different NeoE comPACT proteins, each with a different antigen sequence. [Figure 12]FIG. 12 shows size exclusion chromatography spectra of the eight NeoE comPACT proteins of FIG. 11. [Figure 13A-B] Figure 13A shows the NeoE comPACT protein (linear amplicon) generated using the PCR assembly method described in Figure 6 compared to the NeoE comPACT protein (plasmid) generated from a plasmid. Figure 13B shows a DNA gel of the linear amplicons generated by the PCR assembly method. Each lane contains a comPACT minigene with a different neoepitope sequence. [Figure 14] FIG. 1 shows a streptavidin bead pull-down assay to test for complete biotinylation of comPACT proteins. [Figure 15] FIG. 1 shows biotinylation of different comPACT proteins in crude cell lysates visualized via Western blot using streptavidin-HRP. [Figures 16A-C] FIG. 16 shows the production and purification of BirA enzymes (16B and 16C) and TEV protease (16A) in E. coli. [Figure 17] Figure 1 shows biotinylation of comPACT protein with BirA (lane 2) and cleavage of the His6 tag with TEV protease (lane 3). Untreated comPACT protein is shown in lane 1. [Figure 18] FIG. 1 shows cell sorting of BirA and V5 transduced cells based on V5 expression. [Figure 19] FIG. 1 shows antigen-specific capture of T cells using multimerized comPACT protein. [Figure 20] FIG. 1 shows comPACT NTAmer generation using S88Cβ2M comPACT protein. [Figure 21] Figure 1 shows the coupling of Cy5 to the S88C comPACT protein monomer. [Figure 22A-B]Figure 22A shows an exemplary schematic of the cloning strategy for producing comPACT polynucleotides, and Figure 22B provides sequence validation statistics obtained from 824 individual comPACT polynucleotides. [Figure 23] FIG. 1 shows protein expression and purification for a representative selection of comPACT proteins. [Figure 24] 1 provides an exemplary schematic of the workflow for producing comPACT polynucleotides and proteins. [Figure 25] FIG. 1 shows biotinylation of EBV and MART-1 comPACT proteins with BirA. [Figure 26A-B] FIG. 1 shows the percent of patients covered by the top US HLA I alleles relative to comPACT HLA repertoire size. [Figure 27A] FIG. 1 shows comPACT protein monodispersity, yield, and expression for a representative selection of comPACT proteins. [Figure 27B] FIG. 1 shows comPACT protein monodispersity, yield, and expression for a representative selection of comPACT proteins. [Figure 27C] FIG. 1 shows comPACT protein monodispersity, yield, and expression for a representative selection of comPACT proteins. [Figure 28A-C] FIG. 1 shows that neo12 comPACT molecules modified with Cy5 and His tags bind to neo12 TCR-edited T cells. [Figure 29A-D] FIG. 1 shows that Neol2 NTAmer binds to T cells in an antigen-specific manner and that the presence of imidazole prevents NTAmer binding. [Figure 30A-D] FIG. 1 shows that MART-1 NTAmers bind to T cells in an antigen-specific manner. [Figure 31A-C] FIG. 1 shows that F5 TCR-edited T cells exhibit rapid Cy5 signal decay after NTAmer disruption by imidazole. DETAILED DESCRIPTION OF THE INVENTION

[0067] definition Terms used in the claims and specification are defined as follows, unless otherwise specified.

[0068] Embodiments of the compositions and methods disclosed herein include recombinant antigen-MHC complexes that are capable of pairing with cognate T cells. As used herein, "antigen-MHC," "antigen-MHC complex," "recombinant antigen-MHC complex," "peptide MHC," and "p / MHC" are used interchangeably to refer to a major histocompatibility complex with a peptide within the antigen-binding groove of the MHC.

[0069] As used herein, "antigen" includes any antigen, including patient-specific neoantigens. "Antigenic peptide" refers to a peptide or peptide fragment capable of binding to an MHC molecule. "Neoantigen" refers to an antigen that has at least one alteration, e.g., a mutation in the polypeptide sequence, a difference in post-translational modification, or a difference in expression level, that makes the neoantigen or presentation of the neoantigen different from its corresponding wild-type antigen. "Tumor neoantigen" refers to a neoantigen derived from a tumor or cancer, e.g., a patient's tumor.

[0070] As used herein, "polynucleotide" can refer to ssDNA, dsDNA, ssRNA, dsRNA, or mRNA. Those skilled in the art will understand which form is being referred to based, for example, on the context in which the polynucleotide is being used.

[0071] The term "in vivo" refers to a process that takes place in a living organism, including a cell.

[0072] The term "mammal", as used herein, includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0073] The term "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can be over a region of the sequences being compared, e.g., a functional domain, or alternatively, over the full length of the two sequences to be compared.

[0074] For sequence comparison, typically, one sequence acts as the reference sequence with which test sequence is compared.When using sequence comparison algorithm, test and reference sequence are input into computer, and subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence (s) with reference sequence based on designated program parameters.

[0075] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA; Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0076] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0077] It should be noted that as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0078] Other Interpretation Rules Ranges recited herein are understood to be shorthand for all values ​​within that range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. For example, subranges of 1 to 50 can include 2 to 40, 5 to 25, and 10 to 20.

[0079] Nucleotide and Peptide Compositions T cell-mediated immunity is characterized by the activation of antigen-specific cytotoxic T cells, which can induce death in cells that present antigens in the major histocompatibility complex (MHC) on their surface. These cells that present antigen-loaded MHC complexes include virus-infected cells, cells with intracellular bacteria, cells that have internalized or phagocytosed extracellular sources of protein, and cancer cells that present tumor antigens.

[0080] Native class I MHC heavy chains contain approximately 350 amino acids; native β2-microglobulin contains approximately 100 amino acids; and class I antigenic peptides are typically about 7 to 15 amino acids in length. Class I heavy chains are encoded by major histocompatibility complex genes designated HLA-A, -B, and -C in humans and H-2K, D, and L in mice. Class I heavy chains and β2-microglobulin are encoded separately on different chromosomes. Antigenic peptides are typically processed by cells from protein sources, such as viruses, bacteria, or cancer cells. Numerous variants have been identified for the polypeptides encoded by the HLA-A, -B, and -C MHC genes in humans and the H-2K, D, and L MHC genes in mice.

[0081] Method embodiments disclosed herein relate to producing single molecules in which a selected antigen (e.g., a neoantigen) is linked to an MHC complex comprising β2-microglobulin (β2M) and an MHC heavy chain. Different MHC heavy chains can be linked to the β2M molecule to form a variable number of MHC templates. By utilizing universal target sequences flanking the antigen insertion site, the disclosed methods for inserting antigens into MHC templates via restriction digestion or PCR-based assembly provide the ability to construct libraries of different antigen-MHC complexes in a high-throughput manner that can be personalized to a given patient, for example. These exemplary complexes are encompassed by the term "comPACTs" and can then be linked to particles, barcoded particles, or surfaces for use, for example, in isolating and identifying patient-specific T cell populations targeting patient-specific neoantigens. Methods for linking antigen-MHC complexes and the use of such complexes are disclosed in PCT / US2018 / 021611, filed March 8, 2018, which is incorporated herein by reference in its entirety.

[0082] Peptide-MHC complexes Briefly, as used herein, "comPACTs" refers to a single polypeptide fusion comprising a universal target sequence, an antigenic peptide, a second universal target sequence, β2-microglobulin, and an MHC class I heavy chain comprising, for example, the α1, α2, and α3 domains, forming an MHC display moiety. The MHC display moiety can comprise a recombinant MHC molecule. In certain embodiments, comPACT can comprise a disulfide trap as described in U.S. Patent Application Publication Nos. 2009 / 0117153 and 2008 / 0219947, each of which is incorporated herein by reference. The antigen-MHC complex formed by comPACT results in the presentation of the antigen for recognition by a cognate TCR molecule. In some embodiments, the MHC complex can be an MHC class I (MHC I) complex that pairs with CD8-positive (CD8+) T "killer" cells. In some embodiments, the MHC complex can be an MHC class II (MHC II) complex that pairs with CD4-positive (CD4+) T cells. Each comPACT-encoded MHC allele can be easily exchanged for other MHC I or II alleles, allowing antigenic interrogation of T cells from patients of any MHC haplotype.

[0083] In some embodiments, the comPACT MHC class I heavy chain sequence can include one or more amino acid substitutions, additions, and / or deletions, such as a substitution of tyrosine-84 with a non-aromatic amino acid other than proline. In these embodiments, the amino acid substitution can be an amino acid encoded by the standard genetic code, such as leucine, isoleucine, valine, serine, threonine, alanine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid, cysteine, arginine, serine, or glycine, or can be a modified or unusual amino acid. In one embodiment, the comPACT MHC class I heavy chain sequence includes a substitution of tyrosine-84 with alanine. In another embodiment, the comPACT MHC class I heavy chain sequence includes a substitution of tyrosine-84 with cysteine.

[0084] In some embodiments, the MHC allele lacks the transmembrane domain. In some embodiments, the MHC allele lacks the cytoplasmic domain. In some embodiments, the MHC allele lacks the transmembrane domain and the cytoplasmic domain. In some embodiments, the HLA allele lacks the transmembrane domain. In some embodiments, the HLA allele lacks the cytoplasmic domain. In some embodiments, the HLA allele lacks the transmembrane domain and the cytoplasmic domain.

[0085] Any MHC or HLA allele can be used in the comPACT described herein. Exemplary HLA alleles include, but are not limited to, HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA -A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44: 02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA- B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:0 3, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C *03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01. Any other suitable HLA alleles known in the art can be used in comPACT as described herein.

[0086] β2-microglobulin (β2M) can include recombinant β2M molecules. In some embodiments, the β2M sequence can include one or more amino acid substitutions, additions, and / or deletions, as described above. In one embodiment, the substitution includes a serine-88 to cysteine ​​substitution.

[0087] The amino acid and nucleotide sequences for an exemplary comPACT protein with an hGH signal sequence, a dummy neoantigen insert, an HLA*A02:01 allele, an AviTag peptide, a TEV cleavage site, and a concatenated His tag are shown in Table 1 below. TIFF2024170434000002.tif190170

[0088] In some embodiments, the polynucleotide molecule comprises, from 5' to 3', (i) a promoter sequence, (ii) a first universal target sequence comprising the sequence set forth in SEQ ID NO: 3, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target sequence comprising the sequence set forth in SEQ ID NO: 4, (v) a β2M sequence comprising the sequence set forth in SEQ ID NO: 106, (vi) an MHC allele sequence comprising a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 109-174, (vii) a first affinity tag sequence comprising the sequence set forth in SEQ ID NO: 29, (viii) a protease cleavage site sequence comprising the sequence set forth in SEQ ID NO: 31, and (ix) a second affinity tag sequence comprising the sequence set forth in SEQ ID NO: 35. In some embodiments, the polynucleotide molecule comprises, in the 5' to 3' direction, (i) a promoter sequence, (ii) a first universal target sequence comprising the sequence set forth in SEQ ID NO: 3, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target sequence comprising the sequence set forth in SEQ ID NO: 4, (v) a β2M sequence comprising the sequence set forth in SEQ ID NO: 106, and (vi) an MHC allele sequence comprising a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 109-174. In some embodiments, the polynucleotide molecule comprises, from 5' to 3', (i) a promoter sequence, (ii) a first universal target sequence comprising the sequence set forth in SEQ ID NO: 3, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target sequence comprising the sequence set forth in SEQ ID NO: 4, (v) a β2M sequence, (vi) an MHC allele sequence comprising a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 109-174, (vii) a first affinity tag sequence, (viii) a protease cleavage site sequence, and (ix) a second affinity tag.

[0089] In some embodiments, the polypeptide comprises, from 5' to 3', (i) a promoter peptide comprising the sequence set forth in SEQ ID NO: 2, (ii) a first universal target peptide, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target peptide, (v) a β2M peptide comprising the sequence set forth in SEQ ID NO: 105, and (vi) an MHC peptide comprising the sequence set forth in SEQ ID NOs: 38-103.

[0090] Universal Array The antigenic peptides are generally flanked by universal target sequences or portions thereof. These sequences allow for rapid, high-throughput methods for substituting or inserting nucleotide-encoding antigenic peptides into polynucleotide MHC templates. The universal sequences may contain restriction enzyme recognition sites for restriction digestion-based cloning. Exemplary restriction enzyme recognition sites include, but are not limited to, NotI, BamHI, BlpI, BspEI, BstBI, Xbal, HindIII, EcoRI, ApaI, NotI, and any combination thereof. In certain embodiments, one or more universal target sequences are absent from the engineered genetic material, e.g., to reduce or eliminate off-target effects and / or increase specificity.

[0091] The universal target sequence can be 4 to 100, 4 to 15, 15 to 40, 15 to 35, 15 to 30, 20 to 40, 25 to 40, 30 to 40, 15 to 75, 50 to 100, 50 to 75, 75 to 100, 25 to 50, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 nucleotides in length. The universal sequence can be at least 4, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some embodiments, the universal target sequence is 4 to 8 nucleotides in length, e.g., 4, 5, 6, 7, or 8 nucleotides in length. In other embodiments, the universal target sequence is 25-35 nucleotides in length, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. In other embodiments, the universal target sequence is 35-75 nucleotides in length, e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nucleotides in length. In other embodiments, the universal target sequence is at least about 15 nucleotides in length. In some embodiments, the polynucleotide comprises at least two universal target sequences that are not identical. In some embodiments, the polynucleotide comprises at least two universal target sequences that are identical.

[0092] Exemplary universal target sequences are shown in Table 2. TIFF2024170434000003.tif40170

[0093] In some embodiments, the universal target sequence comprises a polymerase chain reaction (PCR) primer target sequence or primer binding site. Universal primer sequences known in the art can be used in the compositions and methods disclosed herein, or the sequence can be different from the universal primer sequences described above.

[0094] In some embodiments, the first and / or second universal target sequence comprises a restriction enzyme cleavage site.

[0095] Linker In various embodiments, comaPACT can include a first flexible linker interposed between the antigenic peptide segment and the β2-microglobulin segment. Such a linker can extend from the carboxyl terminus of the antigenic peptide segment to the amino terminus of the β2-microglobulin segment, or vice versa. Without being limited by theory, when comPACT is expressed, the linked peptide ligand can fold into the binding groove to produce a functional comPACT protein. In various embodiments, the linker can include at least about 4 amino acids and up to about 20 amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0096] In various embodiments, comA PACT can include a second flexible linker interposed between the β2-microglobulin segment and the MHC heavy chain segment. Such a linker can extend from the carboxyl terminus of the β2-microglobulin segment to the amino terminus of the heavy chain segment, or vice versa. Without being limited by theory, when comA PACT is expressed, the β2-microglobulin and heavy chain can fold into a binding groove, resulting in a molecule that can function to promote T cell expansion. In various embodiments, the linker can include at least about 4 amino acids and up to about 20 amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0097] In various embodiments, comPACT can include a third flexible linker interposed between the MHC heavy chain segment and the purification cluster, such a linker can extend and connect from the carboxyl terminus of the heavy chain segment to the amino terminus of the purification cluster, or vice versa.

[0098] Any suitable flexible linker sequence known in the art can be used. Such linker sequences include, but are not limited to, glycine-serine sequences containing one or more repeating units of the GS, SG, GGGGS (G4S), GGGS (G3S), GSGGS, or GCGGS sequence motif. Multiple consecutive units of the linker sequence motif can be used. In some embodiments, the flexible linker sequence contains 1, 2, 3, 4, 5, 6, 7, 8, or 10 repeating units of a suitable linker sequence.

[0099] The linker may also include a cysteine ​​residue for disulfide bonding between the linker sequence and the MHC allele sequence, such that the cysteine ​​residue forms a disulfide trap. In some embodiments, the linker sequence or the second universal target peptide includes a cysteine ​​residue. In some embodiments, the linker sequence or the second universal target peptide includes a cysteine ​​residue that forms a disulfide bond with the MHC allele.

[0100] signal sequence In various embodiments, comPACT polynucleotides and polypeptides can include a signal sequence, e.g., in the case of a polynucleotide, a signal sequence encoding a signal peptide. The signal sequence can be a secretory signal sequence. Secretory signal sequences direct translated proteins through the secretory pathway of mammalian cells and ensure that the translated protein undergoes cellular quality control. Incorporation of a secretory signal can ensure that comPACT proteins are secreted into the cell culture medium, thereby ensuring that they are uniformly and well-folded and more easily isolated from the culture medium or clarified supernatant.

[0101] In one embodiment, the signal sequence is a signal sequence from human growth hormone (hGH). In another embodiment, the signal sequence is a signal sequence from hIG1 kappa light chain, β2 microglobulin (β2M), or the IL2 signal sequence. Additional signal sequences can also be used, including the signal sequence from β2M (e.g., human β2M), or any other eukaryotic or prokaryotic signal sequence known in the art.

[0102] The signal sequence can be 10-100, 10-20, 10-50, 10-40, 20-40, 20-60, 40-90, 40-60, 45-70, 50-80, 60-90, 55-70, 60-80, 70-80, 50-100, 60-100, 70-100, 80-100, or 90-100 nucleotides in length. The signal peptide can be 3-33 amino acids, 3-10 amino acids, 10-30 amino acids, 10-20 amino acids, 15-30 amino acids, or 20-30 amino acids in length.

[0103] Exemplary signal sequences are shown in Table 3. TIFF2024170434000004.tif104170

[0104] In some embodiments, the signal sequence comprises a sequence comprising the sequence set forth in SEQ ID NO: 1, 24, 26, or 28. In some embodiments, the signal sequence comprises a sequence comprising the sequence set forth in SEQ ID NO: 2, 23, 25, or 27. In one embodiment, the signal sequence encodes a human growth hormone (HGH) signal sequence. In another embodiment, the signal sequence comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the signal sequence comprises the sequence set forth in SEQ ID NO: 2.

[0105] promoter The comPACT polynucleotide composition may further comprise, for example, a promoter for transcription of an mRNA transcript that can be translated by a host cell. The promoter may be prokaryotic or eukaryotic (e.g., mammalian) in origin. Any suitable promoter for gene transcription in a cell may be used, such as EF1α, cytomegalovirus (CMV), or SV40. In some embodiments, the comPACT polynucleotide comprises a CMV promoter.

[0106] In various embodiments, the 5' end of the polynucleotide sequence further comprises a promoter sequence linked to the 5' end of the first universal target. In some embodiments, the promoter sequence is selected from the group consisting of CMV, EF1α, and SV40 promoters. In one embodiment, the promoter sequence is a CMV promoter sequence. In one embodiment, the promoter sequence comprises a sequence comprising the sequence set forth in SEQ ID NO: 37.

[0107] Affinity tags The comPACT polynucleotide composition may further comprise at least one sequence encoding an affinity tag or peptide. In some embodiments, the comPACT polynucleotide comprises at least two affinity tag or peptide sequences.

[0108] Any suitable affinity tag or peptide can be used in a comPACT polynucleotide or polypeptide. Such peptides include, but are not limited to, AviTag, streptavidin-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and Myc-tag. The sequence in the polynucleotide comPACT gene is typically translated into a peptide in the comPACT polypeptide. These epitopes can be used for affinity chromatography purification or quantification of the expressed comPACT polypeptide. For example, a His6 tag can be used to purify comPACT proteins by Ni-NTA affinity chromatography. In some embodiments, the His6 tag can be a concatenated His6 tag containing multiple His6 units, optionally with a linker sequence. The first and second affinity tags can be the same, i.e., both His6 tags or both HA tags, or they can be different tags, i.e., a streptavidin tag and a His6 tag.

[0109] Furthermore, AviTag encodes a biotinylation site recognized by the BirA enzyme. Inclusion of this peptide sequence in a protein allows biotinylation of the sequence via enzymatic modification by BirA. Therefore, comPACT polypeptides containing the AviTag sequence and a His6 tag can be biotinylated and purified via the His6 tag by Ni-NTA affinity chromatography. The purity or quantity of the purified protein can then be assessed by biotin visualization using streptavidin or other avidin reagents.

[0110] In some embodiments, the comPACT polynucleotide comprises an AviTag sequence. In some embodiments, the comPACT polypeptide comprises an AviTag peptide. In some embodiments, the comPACT polynucleotide comprises a His6 sequence. In some embodiments, the comPACT polypeptide comprises a His6 peptide. In some embodiments, the comPACT polypeptide comprises a linked His6 peptide. In some embodiments, the comPACT polynucleotide comprises an AviTag sequence and a His6 sequence. In some embodiments, the comPACT polynucleotide comprises an AviTag sequence and a linked His6 sequence. In some embodiments, the comPACT polypeptide comprises an AviTag peptide and a His6 peptide. In some embodiments, the comPACT polypeptide comprises an AviTag peptide and a His6 peptide.

[0111] In some embodiments, the first and second affinity tags are selected from the group consisting of AviTag, streptavidin-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and Myc-tag.

[0112] In some embodiments, the comPACT polynucleotide comprises a FLAG sequence. In some embodiments, the comPACT polypeptide comprises a FLAG peptide. In some embodiments, the comPACT polynucleotide comprises an HA sequence. In some embodiments, the comPACT polypeptide comprises an HA peptide. In some embodiments, the comPACT polynucleotide comprises a Myc sequence. In some embodiments, the comPACT polypeptide comprises a Myc peptide. In some embodiments, the comPACT polynucleotide comprises a streptavidin sequence. In some embodiments, the comPACT polypeptide comprises a streptavidin peptide. In some embodiments, the comPACT polynucleotide comprises an AviTag sequence and a FLAG sequence. In some embodiments, the comPACT polypeptide comprises an AviTag peptide and a FLAG peptide. In some embodiments, the comPACT polynucleotide comprises an AviTag sequence and an HA sequence. In some embodiments, the comPACT polypeptide comprises an AviTag peptide and an HA peptide. In some embodiments, the comPACT polynucleotide comprises an AviTag sequence and a streptavidin sequence. In some embodiments, the comPACT polypeptide comprises an AviTag peptide and a streptavidin peptide.

[0113] In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a TEV cleavage site, and the second affinity tag encodes a FLAG peptide. In some embodiments, the purification cluster comprises an AviTag peptide, a TEV cleavage site, and a FLAG peptide. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a TEV cleavage site, and the second affinity tag encodes an HA peptide. In some embodiments, the purification cluster comprises an AviTag peptide, a TEV cleavage site, and an HA epitope. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a TEV cleavage site, and the second affinity tag encodes a Myc peptide. In some embodiments, the purification cluster comprises an AviTag peptide, a TEV cleavage site, and a Myc peptide. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a TEV cleavage site, and the second affinity tag encodes a streptavidin peptide, hi some embodiments, the purification cluster comprises an AviTag peptide, a TEV cleavage site, and a streptavidin peptide.

[0114] In some embodiments, the first affinity tag sequence encodes a His peptide. In some embodiments, the first affinity tag sequence encodes a FLAG peptide. In some embodiments, the first affinity tag sequence encodes an HA peptide. In some embodiments, the first affinity tag sequence encodes a Myc peptide. In some embodiments, the first affinity tag sequence encodes a streptavidin peptide. In some embodiments, the purification cluster comprises a His peptide. In some embodiments, the purification cluster comprises a FLAG peptide. In some embodiments, the purification cluster comprises an HA peptide. In some embodiments, the purification cluster comprises a Myc peptide. In some embodiments, the purification cluster comprises a streptavidin peptide.

[0115] Protease cleavage site The comPACT polynucleotide composition may further comprise a protease cleavage site, e.g., a sequence encoding a purification cluster. This cleavage site may be encoded between the first and second affinity tag sequences, allowing for cleavage of the second affinity tag from the comPACT protein after comPACT expression and a round of purification has been completed. Any suitable protease cleavage site known in the art may be used, including, but not limited to, cleavage sites recognized by TEV, thrombin, factor Xa, enteropeptidase, and rhinovirus 3C protease, among others.

[0116] In one embodiment, the protease cleavage site sequence is a TEV cleavage site sequence, a thrombin cleavage site sequence, a factor Xa cleavage site sequence, an enteropeptidase cleavage site sequence, and / or a rhinovirus 3C protease cleavage site sequence. In one embodiment, the protease cleavage site nucleotide sequence encodes a TEV cleavage site. In another embodiment, the comPACT polypeptide comprises a TEV protease cleavage site, ENLYFQG. In one embodiment, the protease cleavage site nucleotide sequence encodes a factor Xa cleavage site. In one embodiment, the protease cleavage site nucleotide sequence encodes a rhinovirus 3C cleavage site. In one embodiment, the protease cleavage site nucleotide sequence encodes an enteropeptidase cleavage site. In one embodiment, the protease cleavage site nucleotide sequence encodes a thrombin cleavage site.

[0117] In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a TEV cleavage site, and the second affinity tag encodes a His6 peptide. In some embodiments, the purification cluster comprises an AviTag epitope, a TEV cleavage site, and a His6 epitope. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a thrombin cleavage site, and the second affinity tag encodes a His6 peptide. In some embodiments, the purification cluster comprises an AviTag epitope, a thrombin cleavage site, and a His6 epitope. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a factor Xa cleavage site, and the second affinity tag encodes a His6 peptide. In some embodiments, the purification cluster comprises an AviTag epitope, a factor Xa cleavage site, and a His6 epitope. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes an enteropeptidase cleavage site, and the second affinity tag encodes a His6 peptide. In some embodiments, the purification cluster comprises an AviTag epitope, an enteropeptidase cleavage site, and a His6 epitope. In some embodiments, the first affinity tag sequence encodes an AviTag peptide, the protease cleavage site sequence encodes a rhinovirus 3C cleavage site, and the second affinity tag encodes a His6 peptide. In some embodiments, the purification cluster comprises an AviTag epitope, a rhinovirus 3C cleavage site, and a His6 epitope.

[0118] PolyA tail The comPACT polynucleotide composition can further comprise a polyadenylation (polyA) tail. Mammalian, eukaryotic, or prokaryotic polyA sequence motifs can be used. This sequence can be included when the comPACT polynucleotide is assembled via PCR for direct transfection into a host cell (e.g., not in the context of an expression construct or vector). Any suitable polyA tail and sequence motif can be used in the comPACT polynucleotide, including, but not limited to, SV40, hGH, bGH, and rbGlob sequences. Such sequences include the RNA sequence motif: AAUAA. In one embodiment, the polyA sequence comprises a bGH polyA sequence. In one embodiment, the polyA sequence is selected from a bGH polyA sequence, an SV40 polyA sequence, an hGH polyA sequence, and an rbGlob polyA sequence. In one embodiment, the polyA sequence comprises an SV40 polyA sequence. In one embodiment, the polyA sequence comprises an hGH polyA sequence. In one embodiment, the polyA sequence comprises an rbGlob polyA sequence.

[0119] Antigen sequence The antigen sequences are 5-100, 5-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 20-40, 20-50, 20-60, 20-70, 20-80, 20-19, 20-100, 20-30, 25-35, 20- The antigen sequence may be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. Antigenic peptides can be 3-50, 3-10, 5-15, 7-15, 5-20, 7-20, 10-15, 10-20, 15-20, 20-25, 20-30, 25-35, 30-40, or 40-50 amino acids in length. Antigenic peptides can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Antigenic peptides can include tumor antigens, neoantigens, tumor neoantigens, viral antigens, bacterial antigens, phosphoantigens, or microbial antigens. In one embodiment, the antigenic peptide is a neoantigen. Antigenic peptides can be selected from patient data to select antigens with one or more somatic mutations. Antigenic peptide prediction can include prediction algorithms and predicting the binding of antigenic peptides or neoantigens to HMC alleles. Antigenic peptide prediction is discussed further below.

[0120] In some embodiments, the nucleotide sequence encoding the antigenic peptide is 20-60, 20-30, 25-35, 20-45, 30-45, 40-60, or 45-60 nucleotides in length. In other embodiments, the nucleotide sequence encoding the antigenic peptide is 20-30 nucleotides in length. In some embodiments, the antigenic peptide is 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the antigenic peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 20 amino acids in length.

[0121] Biotinylation The comPACT proteins described herein can be further biotinylated by any suitable method. One such method utilizes BirA biotin-protein ligase, which is commercially available. A specific amino acid sequence known as the AvtiTag sequence (GLNDIFEAQKIEWHE) is encoded in the protein of interest. BirA ligase, d-biotin, and ATP are added to a reaction mixture containing the protein of interest. BirA covalently ligates biotin to the lysine of the AviTag sequence, thereby biotinylating the protein of interest. The newly biotinylated protein can then be purified and used for downstream applications. Other methods for biotinylating proteins known in the art can also be used.

[0122] In some embodiments, the comPACT protein is biotinylated after protein purification with purified BirA protein. In some embodiments, the comPACT protein is biotinylated in a cell lysate during protein purification with purified BirA protein. Such methods using purified BirA and purified or partially purified comPACT protein are referred to as "in vitro" biotinylation. In some embodiments, the comPACT protein is biotinylated on the cell surface by cell-expressed BirA protein in the cytoplasm during protein production within the cell, or is secreted into the cell culture medium. Such methods using cell-expressed BirA and unpurified comPACT protein are referred to as "in vivo" biotinylation.

[0123] Expression constructs and vectors The comPACT polynucleotide molecule can be inserted into an expression construct or vector, for example, for plasmid and protein production. The expression construct or vector can be a plasmid or a viral vector. Any suitable expression construct or vector known in the art can be used, including bacterial expression plasmids, such as E. coli or Bacillus subtilis plasmids; eukaryotic expression vectors, such as Pichia pastoris expression vectors; or viral vectors, such as lentiviral, vaccinia, or baculoviral vectors. Mammalian expression vectors for use in cultured mammalian cell lines, such as Chinese hamster ovary (CHO), HEK293, Expi293, or any other suitable mammalian cell line, are also contemplated. Furthermore, the expression construct or vector can include a nucleotide barcode. The nucleotide barcode can be unique for each expression construct or vector. In some embodiments, nucleotide sequences encoding a signal sequence, beta-2-microglobulin, and an MHC allele can be ligated into an expression construct or vector with a non-coding or dummy antigen insert. This non-coding antigen insert can then be removed by a suitable cloning technique, such as restriction digestion, and the desired antigen sequence can be inserted via ligation or any other suitable cloning technique.

[0124] In some embodiments, provided herein are libraries comprising two or more different vectors encoding different MHC alleles.

[0125] host cell In another aspect, the present invention provides a host cell comprising the polynucleotide molecule or expression construct described herein. The host cell can be any suitable host cell known in the art, including, but not limited to, bacterial cells such as Escherichia coli or Bacillus subtilis, or eukaryotic host cells such as Chinese hamster ovary (CHO), HEK293, Expi293, HeLa, insect cell lines such as Sf9 or Sf12, or yeast cells such as Pichia pastoris. The host cell can also stably express the biotinylation enzyme BirA. The host cell can be a primary cell or an immortalized cell.

[0126] In some embodiments, the polynucleotide is integrated into the cell genome. In some embodiments, the polynucleotide is extrachromosomal. In some embodiments, the host cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from the group consisting of a stem cell, a tumor cell, an immortalized cell, and a fetal cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell expresses a BirA protein or a fragment thereof.

[0127] Library Libraries containing two or more different comPACT polynucleotide molecules, polypeptide molecules, or particle-bound polypeptide molecules are also contemplated. Libraries can contain between 2 and 1000 molecules. In some embodiments, the library comprises from 2 to 900, from 2 to 800, from 2 to 700, from 2 to 600, from 2 to 500, from 2 to 480, from 2 to 400, from 2 to 300, from 2 to 200, from 2 to 100, from 2 to 50, from 2 to 66, from 2 to 48, from 2 to 30, from 2 to 20, from 2 to 19, from 10 to 1000, from 10 to 900, from 10 to 800, from 10 to 700, from 10 to 600, from 10 to 500, from 10 to 480, from 10 to 400, from 10 to 300, from 10 to 200, from 10 to 100, from 10 to 50, from 10 to 66, from 10 to 48, from 10 to 30, from 10 to 20, from 20 to 1000 , 20~900, 20~800, 20~700, 20~600, 20~500, 20~480, 20~400, 20~300, 20~200, 20~100, 20~50, 20~50, 20~66, 20~48, 20~30, 30~1000, 30~900, 30~800, 30~700, 30~600, 30~500, 30~480, 30~400, 30~300, 30~200, 30~100, 30~50, 30~50, 30~66, 30~48, 30~40, 40~1000, 40~900, 40~800, 40~700, 40~600, 40~500, 40~480, 40~400, 40~300, 40~200, 40~100, 40~60, 40~50, 40~66, 40~48, 50~1000, 50~900, 50~800, 50~700, 50~600, 50~500, 50~480, 50~400, 50~300, 50~200, 50~100, 50~60, 50~66, 60~1000, 60~900, 60~800, 60~700, 60~600, 60~500, 60~ and 480, 60-400, 60-300, 60-200, 60-100, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-480, 70-400, 70-300, 70-200, 70-100, 70-80, 70-90, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-480, 80-400, 80-300, 80-200, or 80-100 polynucleotide or polypeptide molecules.In some embodiments, the library includes 2 to 19, 48 to 480, 48 to 66, 66 to 480, 220 to 240, 40 to 60, 48 to 66, 50 to 70, or 60 to 80 polynucleotide or polypeptide molecules. In some embodiments, the library includes at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 48, 50, 55, 60, 65, 66, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300 , 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 600, 562, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 comPACT polynucleotide or polypeptide molecules. In some embodiments, the library contains 2, 10, 15, 20, 24, 48, 66, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 comPACT polynucleotide or polypeptide molecules. The molecules can be polynucleotides, polypeptides, or polypeptides bound to particles. In some embodiments, two or more polynucleotide or polypeptide molecules have different antigenic peptide sequences. In some embodiments, the two or more molecules have different antigenic peptide sequences and different MHC molecules.

[0128] In some embodiments, the library comprises two or more different polynucleotide molecules, each different polynucleotide molecule comprising (i) a first universal sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is non-identical for each of the two or more polynucleotide molecules, (iii) a second universal target sequence, (iv) a β2M sequence, and (v) an MHC allele sequence. In some embodiments, the MHC allele sequence is non-identical for each of the two or more polynucleotide molecules.

[0129] HLA-A*01:01 HLA-A*02:01 HLA-A*03:01 HLA-A*24:02、HLA-A*30:02、HLA-A*31:01、HLA-A*32:01、HLA- A*33:01、HLA-A*68:01、HLA-A*11:01、HLA-A*23:01、HLA-A*30:01、HLA-A*33:03、HLA-A*25:01*9:HLA-A、26:26:26 HLA-A*68:02、HLA-B*07:02、HLA-B*14:02、HLA-B*18:01、HLA-B*27:02、HLA-B*39:01、HLA-B*40:01、4:4:4B*B* :01、HLA-B*50:01、HLA-B*57:01、HLA-B*58:01、HLA-B*08:01、HLA-B*15:01、HLA-B*15:03、HLA-B*35:02-B、HL*35:02-B、HL B*42:01、HLA-B*44:03、HLA-B*51:01、HLA-B*53:01、HLA-B*13:02、HLA-B*15:07、HLA-B*27:05、HLA-17-B、35:35: HLA-B*38:01、HLA-B*41:02、HLA-B*44:05、HLA-B*49:01、HLA-B*52:01、HLA-B*55:01、HLA-C*02:02、3:0A-4C* :01、HLA-C*07:01、HLA-C*01:02、HLA-C*04:01、HLA-C*06:02、HLA-C*07:02、HLA-C*16:01、HLA-C*03:04-AHL、HL C*08:01、HLA-C*08:02、HLA-C*12:02、HLA-C*12:03、HLA-C*14 :02 HLA-C*15:02 and HLA-C*17:01 were also reported in Figure 2 .In one embodiment, the library includes at least the HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01 alleles.

[0130] In some embodiments, the library comprises two or more different polypeptide molecules, wherein the antigenic peptide is not identical for each of the two or more polypeptide molecules, and each different polypeptide is bound to a particle. In some embodiments, the library further comprises a unique, defined barcode sequence operably associated with the identity of each different polypeptide.

[0131] Embodiments can include barcoded polynucleotides containing defined barcode sequences. The barcoded polynucleotides can be polynucleotides that provide a unique antigen-specific sequence for identification after T cell isolation. Thus, each unique comPACT is bound to a particle with a uniquely defined barcode sequence. This allows for an operable association between a given antigen and a given barcode that is unique to that pair.

[0132] The barcoded polynucleotide can be ssDNA or dsDNA. The barcode-containing polynucleotide can be modified at its 5' end to include a binding moiety for binding to particles. For example, a polynucleotide containing a barcode sequence can be conjugated to a biotin molecule to bind to a streptavidin-core attached to particles such as dextran. However, any suitable binding moiety can be used to bind a polynucleotide to particles. As described herein and understood by those skilled in the art, suitable binding moiety pairs are known in the art. Non-limiting examples of binding moieties include thiol, maleimide, adamantane, cyclodextrin, amine, carboxy, azide, and alkyne.

[0133] particle As used herein, "nanoparticle" or alternatively "particle" refers to a substrate that can be specifically sorted or isolated and to which other entities can be bound. In some embodiments, the nanoparticles are magnetic, e.g., for isolation using a magnet. In some embodiments, the magnetic nanoparticles comprise magnetic iron oxide. Examples of magnetic particles include, but are not limited to, Dynabeads™ (Thermo Fisher). In some embodiments, the nanoparticles are polystyrene particles, e.g., for isolation by gravity. In other embodiments, the particles can be surfaces, beads, or polymers. Examples of beads include, but are not limited to, agarose beads and sepharose beads. In certain embodiments, the particles or nanoparticles can be fluorescent or directly or indirectly bound to a fluorophore.

[0134] According to certain embodiments, nanoparticles are modified with binding moieties for attaching additional molecules. The nanoparticle modifications include binding moieties that can pair (e.g., covalently bond) with corresponding cognate (e.g., complementary) binding moieties attached to polynucleotides. Any suitable pair of binding moieties can be used to modify nanoparticles and polynucleotide detection tags for attachment. Non-limiting examples of binding moiety pairs include streptavidin / biotin systems, thiol groups (e.g., cysteine) and maleimides, adamantane and cyclodextrins, amino groups and carboxy groups, and azide groups and alkyne groups. In some embodiments, the binding moiety can include a cleavage moiety. In other embodiments, the binding moiety attached to the complementary cognate binding moiety can be reversible, e.g., a reducible thiol group. In an exemplary embodiment, the modified nanoparticles are streptavidin-coated magnetic nanoparticles, such as 1 μm nanoparticles (e.g., Dynabeads™ MyOne™ Streptavidin T1 beads from ThermoFisher Scientific), and the polynucleotides can be biotinylated for binding to the modified nanoparticles.

[0135] The particles can be dextran, such as biotinylated dextran or streptavidin-coated dextran. Modified dextrans are described in more detail in Bethune et al., BioTechniques 62:123-130, March 2017, and U.S. Patent Application Publication No. 2015 / 0329617, which are incorporated herein by reference in their entirety. Biotinylated comPACT can be bound to streptavidin-coated dextran.

[0136] comPACT can also be assembled into tetramers containing one, two, three, or four biotinylated comPACT proteins bound to a streptavidin core. The tetramers can also contain a fluorophore, such as phycoerythrin (PE) or allophycocyanin (APC), bound to the streptavidin core. MHC class I and II tetramers are well known in the art. MHC class I tetramers are described in further detail in Burrow SR et al., J Immunol, 2000, December 1, 165(11), pp. 6229-6234, and MHC class II tetramers are described in further detail in Nepom GT, J Immunol, 2012, March 15, 188(6), pp. 2477-2482, both of which are incorporated herein by reference in their entireties.

[0137] ComPACT proteins can also be assembled into multimers. In some embodiments, the comPACT protein multimer can be a dimer, trimer, tetramer, pentamer, hexamer, or higher multimer. In some embodiments, the multimer can contain at least two or more comPACT proteins. In some embodiments, the multimer can contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 comPACT proteins.

[0138] Generation method Antigen prediction To produce comPACT, one of the initial steps can involve identifying a patient's tumor-specific antigens (e.g., neoantigens). Compositions generated by this method can then be utilized in T cell-mediated immune processes, such as patient-specific cancer immunotherapy. To identify a patient's putative neoantigens (tumor or pathogen), an in silico prediction algorithm program can be used to analyze tumor, viral, or bacterial sequencing data, including whole-genome, whole-exome, or transcriptome sequencing data, to identify one or more mutations corresponding to putatively expressed neoantigens. Furthermore, human leukocyte antigen (HLA) typing can be determined from the patient's tumor or blood sample, and this HLA information can be used together with the identified putative neoantigen peptide sequence in a predictive algorithm for MHC binding, as validated by Fritsch et al., 2014, Cancer Immunol Res., 2:522-529, the entire contents of which are incorporated herein by reference. Common HLAs found in human populations can also be included in neoantigen prediction algorithms, such as HLA-A*02, 24, 01; HLA-B*35, 44, 51; DRB1*11, 13, 07 in Caucasians; HLA-A*02, 03, 30; HLA-B*35, 15, 44; DRB1*13, 11, 03 in African-Brazilians; and HLA-A*24, 02, 26; HLA-B*40, 51, 52; DRB1*04, 15, 09 in Asians. Specific pairings of HLA alleles can also be used. Common alleles found in human populations are further described in Bardi et al. (Rev Bras Hematol Hemoter. 2012; 34(1):25-30).

[0139] Further examples of methods for identifying neoantigens include combining sequencing with mass spectrometry and MHC presentation prediction (e.g., U.S. Patent Application Publication No. 2017 / 0199961) and combining sequencing with MHC binding affinity prediction (e.g., issued U.S. Patent No. 9,115,402). Additionally, methods useful for identifying the presence of neoantigen-specific T cells in a patient sample can be used in combination with the methods described herein, such as those described in U.S. Patent Application Publication Nos. 2017 / 0003288 and PCT / US17 / 59598, which are incorporated by reference in their entireties. These analyses result in a ranked list of candidate neoantigen peptides for the patient that can be readily synthesized using routine methods for screening cognate antigen-specific T cells.

[0140] Primer annealing and restriction digest assembly In general, preparation of comaPACT polynucleotides can be accomplished by the procedures disclosed herein and by recognized recombinant DNA techniques, such as preparation of plasmid DNA, restriction enzyme digestion of DNA, DNA ligation, transformation or transfection of the host, culture of the host, and isolation and purification of the expressed fusion complex. Such procedures are generally known and are disclosed in standard works such as Sambrook et al. (supra).

[0141] In some embodiments, DNA encoding an MHC class I heavy chain can be obtained from a suitable cell line, such as, for example, a human lymphoblastoid cell. In various configurations, the gene or cDNA encoding the class I heavy chain can be amplified by polymerase chain reaction (PCR) or other means known in the art. In some embodiments, the PCR product can also include a sequence encoding a linker and / or one or more restriction enzyme sites for ligating such a sequence.

[0142] In some embodiments, vectors encoding comPACT polynucleotides can be prepared by ligating sequences encoding MHC heavy chains and β2-microglobulin to sequences encoding antigenic peptides. DNA encoding antigenic peptides can be obtained by isolating DNA from natural sources or by known synthetic methods, such as the phosphotriester method. See, for example, Oligonucleotide Synthesis, IRL Press (M. Gait, ed., 1984). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. A DNA sequence encoding a universal target sequence discussed herein can be interposed between the sequence encoding a signal sequence and the sequence encoding the antigenic peptide, and a second universal target sequence can be interposed between the sequence encoding the antigenic peptide segment and the sequence encoding the β2-microglobulin segment. In some embodiments, the segments can be ligated using a ligase. In some embodiments, the sequence encoding the antigenic peptide can be phosphorylated with an appropriate polynucleotide kinase. In some embodiments, the polynucleotide kinase is T4 polynucleotide kinase. Any suitable polynucleotide kinase known in the art can be used, including but not limited to T4 polynucleotide kinase, also known as T7 polynucleotide kinase.

[0143] PCR assembly In some embodiments, comPACT can be assembled via polymerase chain reaction (PCR) amplification. Similar to the restriction digestion method, DNA encoding the MHC heavy chain and β2-microglobulin can be obtained from a suitable source. A second DNA fragment encoding a selected signal sequence can also be obtained from a suitable source. Both DNA fragments can have different universal target sequences, so that a primer for one universal sequence will not anneal to the second universal sequence. Two sequences encoding a selected antigen peptide can be synthesized: one forward primer with the antigen sequence at its 5' end and the complement of the universal primer sequence on the MHC DNA fragment at its 3' end; and one reverse primer with the reverse complement of the selected antigen sequence at its 5' end and the reverse complement of the universal primer from the signal sequence fragment at its 3' end. PCR reaction with all four DNA fragments and primers for the 5' end of the signal sequence fragment and the 3' end of the MHC allele fragment results in the amplification of two DNA fragments: a fragment with the signal sequence at its 3' end and the antigen sequence at its 5' end, and a fragment with the antigen sequence at its 3' end and the MHC allele at its 3' end. Further PCR amplification cycles allow the overlapping antigen peptide sequences to anneal, resulting in a single full-length DNA fragment. In some embodiments, the signal peptide fragment further comprises a promoter sequence. In some embodiments, the MHC fragment further comprises a purification cluster and / or a polyA tail.

[0144] Transfection, transduction, and genetic modification of host cells comPACT polynucleotides can be inserted into host cells via known suitable methods, including but not limited to, transfection, transduction, electroporation, lipofection, sonoporation, mechanical disruption, or viral vectors. Exemplary transfection reagents include, but are not limited to, Expifectamine, Lipofectamine, polyethyleneimine (PEI), or Fugene. In some instances, Expifectamine is used to transfect mammalian cells with comPACT polynucleotides.

[0145] comPACT polynucleotide can be transiently or stably expressed in host cell.In some embodiments, comPACT polynucleotide is integrated into host genome.In other embodiments, comPACT polynucleotide remains extrachromosomal.Can also use any suitable gene editing technology known in the art to modify host cell with comPACT polynucleotide, including CRISPR / Cas9, zinc finger nuclease or TALEN nuclease.

[0146] Expression Numerous strategies can be used to express the comPACT polyprotein. For example, comPACT can be incorporated into a suitable vector by known methods, such as by using restriction enzymes and ligases (see, e.g., Sambrook et al., supra). The vector can be selected based on factors related to the cloning protocol. For example, the vector can be compatible with the host cell being employed and have an appropriate replicon. Suitable host cells can include eukaryotic and prokaryotic cells that are easily transformed and exhibit rapid growth in culture media. Examples of host cells include prokaryotes such as Escherichia coli and Bacillus subtilis, and eukaryotes such as animal cells and yeast, e.g., mammalian and human cells. Non-limiting examples of mammalian cells that can be used as hosts for expressing comPACT include J558, NSO, SP2-O, 293T, Expi293, and CHO. Other examples of possible hosts include insect cells such as Sf9 or Sf12, which can be grown using conventional culture conditions. See Sambrook et al., supra. In various embodiments, cells expressing comPACT polypeptides can be identified using known methods, for example, expression of comPACT polypeptides can be determined by ELISA or Western blot using an antibody probe against the MHC heavy chain portion of comPACT, or an antibody against an affinity tag such as His6, or streptavidin reagent if comPACT is biotinylated.

[0147] In some embodiments, comPACT is expressed in mammalian cells. The advantages of expressing proteins in mammalian cells rather than E. coli cells are manifold. Proteins expressed in E. coli cells must be carefully purified from lipopolysaccharide (LPS). Expression of proteins in mammalian cells does not result in LPS contamination of the purified protein. Furthermore, because mammalian cells produce proteins with the correct post-translational modifications required for proper folding, including proper formation of disulfide bonds, mammalian cells are more likely to properly fold mammalian proteins. Furthermore, mammalian cells provide the correct chaperone proteins to assist protein folding in the endoplasmic reticulum or Golgi apparatus. This results in increased purification of uniformly well-folded proteins compared to proteins expressed in E. coli cells.

[0148] comPACT can be substantially free of LPS. comPACT can be free of LPS, e.g., comPACT can have no detectable LPS as measured using LPS detection methods known in the art. comPACT can be glycosylated. comPACT can be modified via expression in eukaryotic or mammalian cells, e.g., via one or more post-translational modifications, such as glycosylation. comPACT can include one or more post-translational modifications. comPACT can be (1) substantially free of or free of LPS; and (2) glycosylated.

[0149] Example ComPACT Workflow Process Figure 24 shows an exemplary schematic diagram of comPACT protein assembly and expression. Sense and antisense oligos encoding the desired neo-antigen peptide sequence are synthesized and annealed to form double-stranded oligos with overhangs at the 5' and 3' ends. These oligos can then be ligated into a plasmid containing the β2M gene and MHC alleles. The complete comPACT oligos can be amplified into double-stranded amplicons and transfected into cells for protein expression and optional biotinylation. comPACT proteins can be assessed via SDS-PAGE. ComPACTs can then be selected for scale-up of plasmid production in E. coli. Protein-producing cells are transfected with the selected plasmids, and comPACTs are purified from the producing cells for use in functional assays.

[0150] Purification (chromatography) The expressed comPACT polypeptide can be isolated and purified by known methods. For example, comPACT containing a His6 affinity tag can be purified by affinity chromatography on a Ni-NTA column by procedures that are generally known and disclosed. Furthermore, comPACT containing human HLA sequences can be purified by affinity chromatography on a monoclonal antibody-Sepharose column by procedures that are generally known and disclosed.

[0151] Isolation of T cells In another aspect, provided herein is a method for isolating antigen-specific T cells, the method comprising: (a) providing a polypeptide comprising, from amino terminus to carboxyl terminus, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide, wherein the polypeptide is linked to a particle; (b) providing a sample known or suspected to contain one or more T cells; (c) contacting the polypeptide with the sample, comprising providing conditions sufficient for a single T cell to bind to the polypeptide bound to the particle; and (d) isolating a single T cell bound to the particle.

[0152] Isolation and identification of patient-derived and antigen-specific T cells using comPACT as described herein can include incubating comPACT proteins with patient-derived T cells. In some embodiments, a library containing at least two comPACT proteins can be incubated with patient-derived T cells. T cells can be prepared using standard methods starting from tissues such as blood, lymph nodes, or tumors.

[0153] Patient-derived T cells can be isolated from the patient's peripheral blood mononuclear cells (PBMCs) or tumor-infiltrating lymphocytes (TILs). For example, both CD4+ and CD8+ T cells can be labeled and sorted from PBMCs or TILs with anti-CD4 and anti-CD8 fluorescent antibodies to isolate only CD4+ or CD8+ cells, with the live population of CD4+ and CD8+ single-positive cells sorted using fluorescence-activated cell sorting (FACS). In some embodiments, T cells positive for both CD4 and CD8 can be isolated using anti-CD3 fluorescent antibodies followed by FACS. Those skilled in the art can determine the type of T cells to isolate based on the type or types of comPACT protein used.

[0154] Incubating comPACT or a comPACT library with a T cell suspension allows the particle-bound antigen to be fully and thoroughly exposed to various T cell receptors. This method may include rocking or rolling the cells. In some embodiments, comPACT binds to the particles.

[0155] After incubation of comPACT or a comPACT library with T cells, the bound comPACT-T cell complexes are selectively sorted or recovered. T cells are likely to bind to multiple identical copies of the same comPACT library element and can be sorted based on these interactions. For example, if comPACT contains a fluorophore or is coupled to particles bearing a fluorophore, T cells can be selectively isolated using fluorescence-associated cell sorting (FACS), including single-cell sorting. If comPACT is coupled to magnetic particles, a magnet can be applied to the suspension to separate particles complexed with antigen-paired T cells and remove unpaired T cells. Alternatively, if the particles are polystyrene particles, unpaired T cells can be separated by gravity (e.g., centrifugation). After removal of unpaired T cells, in some embodiments, the sorted bound particles are washed at least once to remove nonspecifically bound T cells.

[0156] ComPACT-bound T cells can also be sorted by FACS into individual collection vessels, such as multiwell plates. Individual collection vessels can be single-cell reactors. For example, components used for downstream processing and analysis can be added to each single-cell reactor. comPACT-bound T cells can also be sorted by FACS into bulk collection vessels (e.g., all isolated T cells are collected in the same vessel).

[0157] ComPACT-bound T cells can also be individually isolated in droplets using a droplet generating microfluidic device (i.e., a "droplet generator"). Droplet generating devices used to encapsulate single cells are known to those of skill in the art and are described, for example, in U.S. Application Publication No. 2006 / 0079583, U.S. Application Publication No. 2006 / 0079584, U.S. Application Publication No. 2010 / 0021984, U.S. Application Publication No. 2015 / 0376609, U.S. Application Publication No. 2009 / 0235990, and U.S. Application Publication No. 2004 / 0180346.

[0158] After isolating comPACT-bound T cells into single-cell reactors (e.g., isolated in individual wells or droplets), the nucleic acids of the comPACT-bound T cells can be further processed for downstream analysis. Specifically, expressed TCRα and TCRβ mRNA transcripts can first be converted to cDNA by reverse transcription, and the cDNA is amplified for next-generation sequencing (NGS) methods known to those skilled in the art, including, but not limited to, sequencing by synthesis (Illumina). [Example]

[0159] Example 1: Design and cloning of comPACT minigenes by restriction enzyme digestion cloning Construction of comPACT minigene for restriction enzyme digestion: The basic exemplary components of a comPACT minigene are a signal sequence directing protein secretion, a universal target sequence such as a restriction enzyme recognition site or primer binding site, an antigenic peptide (or neoantigen, NeoE), a second universal target site, an invariant β2M, the extracellular domain of an MHC allele, and a purification cluster that enables comPACT purification via enzymatic modification (e.g., biotinylation) and affinity tags. The cluster may also contain a protease cleavage site and a linker sequence between the peptide components. The minigene may also contain cysteine ​​mutations that act as disulfide traps. A schematic diagram of the comPACT minigene is shown in Figure 1. Additional restriction enzyme recognition sites upstream and downstream of the MHC heavy chain sequence can be used to insert other MHC alleles to construct different MHC templates and construct libraries of MHC templates (Figure 2). The DNA fragment encoding the signal sequence, universal target sequence, invariant β2M, and the extracellular domain of an MHC allele is the base MHC template.

[0160] For the restriction digestion cloning method, each comPACT DNA construct is a base MHC template with three in-frame stop codons and a dummy antigen sequence insert containing unique restriction enzyme recognition sites for destruction of the uncut or religated template (Figure 3). This construct can be used as part of a ready-to-use platform for rapidly assembling libraries of antigenic peptides complexed with their MHC alleles. Additionally, MHC alleles can be modified or mutated (e.g., Y84A or Y84C) to improve folding or increase binding of antigenic peptides to the MHC protein. Furthermore, β2M proteins can also be mutated (e.g., S88C) and conjugated with thiol dyes.

[0161] In this example, a comPACT minigene is shown having the following structure: a NotI restriction enzyme recognition site at the 5' end; a signal sequence from human growth hormone hGH shown in Table 3; a restriction enzyme recognition site Blp1 upstream of the antigenic peptide region and a BamHI restriction enzyme recognition site downstream of the antigenic peptide sequence shown in Table 2; a linker sequence of primarily glycine and serine residues (i.e., a Gly-Ser linker); a β2M sequence; a second Gly-Ser linker sequence with a BspI restriction enzyme recognition site; an MHC heavy chain; a third Gly-Ser linker sequence with a BstBI restriction enzyme recognition site; and a purification cluster with an AviTag sequence, a TEV cleavage site, and a linked histidine tag.

[0162] Restriction digest cloning and assembly of comPACT minigenes Three different methods for inserting neoantigens via restriction digestion are described herein. In the first, as shown diagrammatically in Figure 4, a primer encoding the antigenic peptide (NeoE) spans a first restriction enzyme recognition site (BlpI in this example) at the 5' end and a second restriction enzyme recognition site (BamHI in this example) at the 3' end. This primer amplifies a universal reverse primer encoding the second restriction enzyme recognition site, generating a primer-dimer of approximately 70 bp.

[0163] In the second method, a primer encoding the antigenic peptide extends a second restriction enzyme recognition site at its 5' end, which is the reverse complement of the antigen. This primer primes in the reverse direction from the template DNA encoding the signal sequence. When paired with a forward primer that extends into the first restriction enzyme recognition site sequence, the reaction generates a 70 bp product, or approximately 140 bp if a forward primer that extends into a restriction enzyme recognition site further upstream of the antigenic site is used.

[0164] In both the first and second methods, the insert is washed on a commercially available column, digested with the appropriate restriction enzyme, washed again on a commercially available column, and then ligated with a predigested MHC template in a vector. The ligation reaction is transformed into E. coli, and the plasmid prepared from the transformed E. coli is used in a mammalian producer cell transfection reaction.

[0165] Example 2: Design and cloning of comPACT minigenes via primer annealing In a third variant of the MHC template-vector ligation, PCR and restriction digestion were bypassed by annealing two reverse-complementary neoantigen-encoding primers. These primers were designed to have 5' and 3' ends beginning and ending with complementary sequences simulating overhangs from restriction digestion (Figure 5). The sense and antisense primers were incubated with T4 polynucleotide kinase and ATP to phosphorylate the 5' ends (Figure 22A). When these primers annealed to each other, a double-stranded oligonucleotide sequence was formed, with overhanging nucleotides as if digested with a restriction enzyme. The phosphorylated neoantigen insert was ligated into the pre-cleaved MHC template in the vector. The comPACT minigene had the same structure as that described in Example 1. The ligation product was then used for PCR amplification of the linear comPACT amplicon using flanking universal primers to amplify and sequence the complete comPACT gene. Using this method, 824 comPACTs with unique neoantigen sequences were generated, and more than 99% of the generated comPACTs had the correct neoantigen sequence (Figure 22B).

[0166] The ligation product plasmid was then transformed into E. coli and plated onto selective agar plates containing ampicillin. Individual colonies were extracted, grown overnight for plasmid purification, and sequenced for complete gene verification. After sequence confirmation, the plasmid lot was stored and propagated in large quantities.

[0167] Alternatively, if the pre-cleaved MHC template vector retains a 5' phosphate group at its overhanging end, T4 kinase is not used. The annealed antigen insert can then be ligated with the cleaved MHC vector, and the ligation product can be transformed into E. coli for plasmid generation.

[0168] Example 3: Design and cloning of comPACT minigenes via PCR assembly Construction of comPACT minigenes for PCR assembly: A fourth method for inserting neoantigens can also be used. In this method, neoantigens are inserted via polymerase chain reaction into an MHC template flanked by an upstream promoter and a downstream polyadenylation signal, forming a 2.5 kb minigene. A schematic diagram of the PCR assembly reaction is shown in Figure 6.

[0169] In this example, a comPACT minigene is shown with the following structure: a promoter at the 5' end; a signal sequence with a first universal target sequence; an antigenic peptide; a second universal target sequence with a linker sequence containing primarily glycine and serine residues (i.e., a GlySer linker); a β2M sequence; a second Gly-Ser linker sequence; an MHC heavy chain allele; a third Gly-Ser linker sequence; a purification cluster; and a polyA sequence. In this method, the universal target sequences are not the same.

[0170] comPACT minigene PCR assembly: In this method, two primers (<60 nt) containing the selected neo-antigen sequence are synthesized. The first primer contains the neo-antigen sequence at its 5' end, followed by a stretch of the second universal target sequence at its 3' end. The second primer contains the reverse complement of the neo-antigen sequence at its 5' end and the reverse complement of the first universal target sequence at its 3' end. These primers are mixed with a DNA fragment encoding the promoter region, signal sequence, and first universal target sequence, as well as another DNA fragment encoding the second universal target sequence, β2M sequence, MHC allele, purification cluster, and polyA sequence. Each antigen peptide primer anneals to its complementary sequence, and a PCR reaction is performed to amplify the neo-antigen sequence in either the promoter fragment or the MHC allele fragment. These two newly synthesized fragments each contain the neo-antigen sequence. A further PCR reaction, along with primers at the 5' end of the promoter sequence and the 3' end of the polyA sequence, allows the neo-antigen sequences to anneal to each other and prime the assembly of a full-length linear comPACT amplicon.

[0171] The fully assembled linear comPACT polynucleotide is then cleaned up for direct transfection into mammalian production cells, bypassing steps using E. coli and plasmid production entirely.

[0172] Example 4: Expression and purification of comPACT protein from a plasmid Protein expression Neoantigen 12 (neo12) was ligated to the HLA-A2 template sequence and inserted into an expression plasmid (pPACT0010) via restriction digestion of NotI and BamHI restriction enzyme recognition sites and ligation as previously described.

[0173] Expi293 mammalian production cells in a 30 mL shake flask were transfected with pPACT0010 incubated with Expifectamine transfection reagent on day -1. The enhancer included in the Expifectamine transfection kit was added on day 0. Samples were collected from cell supernatants on days 1–7 and assessed for secreted protein via SDS-PAGE and total protein staining using Safestain (ThermoFisher). Secreted comPACT protein levels increased until day 3, at which point protein secretion plateaued (Figure 7). Secreted comPACT protein was initially identified by its apparent molecular weight (=53 kDa) and confirmed by Western blot using NTA-HRP to detect the His6 affinity tag.

[0174] Protein purification Neo12 comPACT protein harvested on day 7 was purified by Ni-NTA affinity chromatography via binding of the His6 affinity tag. Samples were assayed for total protein via SDS-PAGE and Safestain. The absence of comPACT protein in the affinity column flow-through (FT) fraction confirmed that the His6 tag was not cleaved during expression and purification (Figure 8). Purification yields exceeded 400 mg / L culture volume.

[0175] The Neo12 comPACT protein was biotinylated (discussed in Example 6 below) and further purified by size-exclusion chromatography. A single major peak was observed, suggesting that the protein was properly folded, monomeric, and had little aggregation (Figure 9). The second peak was ATP added for the BirA-catalyzed biotinylation reaction.

[0176] Optimized production volume and parallel production comPACT production was scaled down from a culture volume of 30 mL in shake flasks to 0.7 mL in a 96-deep-well shake block. Expi293 mammalian production cells were transfected with plasmid DNA containing the pPACT0010 plasmid, and secreted Neo12 comPACT protein was purified as previously described. 437 mg / L of purified Neo12 comPACT protein was recovered from a 0.7 mL well volume, compared to the previously reported yield of over 400 mg / L from a 30 mL purification experiment (Figure 10). The protein yield from the 0.7 mL experiment corresponds to over 300 μg of protein, or approximately 1000-fold higher than what is generally required for a typical flow cytometry experiment.

[0177] Next, we evaluated the parallel expression of multiple comPACT constructs. Eight different comPACT constructs carrying different neoantigens (neoantigens 10, 15, 64, 65, 66, 67, 80, and 83) were expressed in 30 mL shake flasks as an intermediate-processing assay (Figure 11). Each comPACT construct was transfected into cells as described above, and comPACT proteins were expressed and secreted into the cell supernatant. The expressed proteins were purified, concentrated, and normalized as described above. Samples of the crude supernatant and concentrated protein were assayed for total protein as described above. comPACT proteins were purified by size-exclusion chromatography (Figure 12). A single peak containing 2–20 mg of protein was observed for each protein, suggesting that the comPACT proteins were properly folded and monomeric.

[0178] Example 5: Expression and purification of comPACT protein from linear amplicons In the previous example, comPACT proteins were expressed from plasmids transfected into mammalian producer cells. Alternatively, a linear amplicon of the neo12 comPACT minigene (neoantigen 12 assembled into a minigene with the HLA-A2 template sequence) flanked by promoter and polyA sequences was transfected into 0.7 mL of producer cells in a 96-deep-well plate. As a control, the pPACT0010 plasmid was also transfected into separate producer cells. Proteins from both samples were expressed, purified, and assayed for total protein as described above. Similar levels of expressed protein were produced by both the linear amplicon and the plasmid (Figure 13A), suggesting that proteins encoded by comPACT minigenes can be produced without the use of a plasmid intermediate. Several different comPACT minigenes with different neoepitope sequences were generated for direct transfection of producer cells (Figure 13B).

[0179] Additional comPACT clones with different HLA alleles were generated using the annealing and phosphorylation workflow described in Example 2. Linear amplicons were derived from expression vectors using flanked PCR and universal primers and transfected into Expi293F cells for comPACT protein production. Figure 23 shows a protein gel of representative comPACT proteins generated using the annealing cloning workflow. ComPACT proteins were run on an SDS-PAGE gel and stained with Safestain (Thermofisher).

[0180] Example 6: Biotinylation of comPACT proteins In vitro biotinylation of comPACTs by isolated BirA enzyme The comPACT-purified cluster contains the BirA recognition sequence (Avitag) for biotinylation. Purified comPACT protein was either unbiotinylated (untreated with BirA) or biotinylated with commercially available BirA protein according to the manufacturer's instructions (treated with BirA). After overnight BirA enzyme treatment, the samples were bound to two types of magnetic streptavidin beads (C1 and T1) and incubated to allow the biotinylated protein to bind to the streptavidin beads. The supernatant (SN) and beads ("pellet," P) were fractionated via SDS-PAGE. Samples were assayed for the presence of total protein and comPACT protein via Western blot with NTA-HRP using Safestain (Figure 14). In untreated samples, comPACT protein was found primarily in the SN fraction, confirming its lack of biotinylation. Although the interaction between the biotinylated protein and the C1 streptavidin beads was most complete in the biotinylated sample, comPACT protein was found in the pellet samples of both the C1 and T1 streptavidin beads. Biotinylated comPACT protein was not detected via Western blot in the C1 streptavidin bead-cleared supernatant, suggesting that approximately 100% of the comPACT protein was biotinylated.

[0181] ComPACT proteins can also be biotinylated in the clarified supernatant prior to purification. Multiple comPACT proteins were expressed in production cells as described above. Cell culture supernatants were collected and clarified by centrifugation. The clarified supernatants were treated with commercially available BirA protein according to the manufacturer's instructions and then purified via Ni-NTA affinity chromatography. Biotinylation was assessed via Western blot (Figure 15). All comPACT proteins tested were biotinylated using this method, demonstrating effective biotinylation of comPACT proteins in clarified cell supernatants.

[0182] To generate sufficient BirA for high-throughput biotinylation of comPACT proteins, a His6-tagged BirA protein was expressed in E. coli cells. This His6-tagged BirA was purified by Ni-NTA affinity chromatography (Figure 16B) and used to biotinylate comPACT proteins. A second version, BirA-His6, with a TEV-cleavable His6 tag was also expressed and purified by Ni-NTA affinity chromatography (Figure 16C). This BirA-TEV-His6 protein can be purified via Ni-NTA, the His6 tag removed via TEV cleavage, and then the untagged BirA is used to biotinylate comPACT proteins. After biotinylation of comPACT proteins, the untagged BirA protein can be purified by Ni-NTA affinity chromatography. Additionally, TEV protease was heterologously expressed in E. coli for use with BirA-TEV-His6 (Figure 16A) for use in generating biotinylated comPACT protein.

[0183] Cleavage of the His6 tag on the comPACT protein after biotinylation was also evaluated, and the results are shown in Figure 17. ComPACT proteins were either treated with or without BirA, and then biotinylated as described above (lanes 1 and 2 in Figure 17). A third sample of comPACT protein was treated with BirA and then with TEV to cleave the His6 tag present on the protein (lane 3). The samples were fractionated via SDS-PAGE, and total protein was assessed via Safestain. All three samples contained equal amounts of comPACT protein. Biotinylation of the comPACT protein and cleavage of the His6 tag were assessed by Western blot using SA-HRP reagent for the biotin signal and NTA-HRP reagent for the His6 tag. The non-biotinylated sample showed no biotin signal but contained a His6 signal, while the biotinylated and undesorbed samples contained both signals. The biotinylated and TEV-cleaved sample had only a biotin signal, indicating that the His6 tag was successfully cleaved from the comPACT protein.

[0184] In vivo biotinylation of comPACT in cells expressing BirA A third approach to biotinylating comPACTs is to express BirA in Expi293-generating cells prior to purification and biotinylate comPACTs in vivo. Expi293 cells were transduced with a lentiviral vector to coexpress BirA flanked by V5, which acts as a cell surface transduction marker. Transduced cells sorted for V5 also express BirA (Figure 18). These cells can be used to generate biotinylated comPACTs in vivo prior to comPACT protein purification. Two cell lines were generated that either secrete BirA into the medium (Expi293v0223) or express BirA on the cell surface (Expi293v0263). A third cell line was also generated that expresses BirA flanked by the endoplasmic reticulum (ER) retention sequence KDEL.

[0185] To evaluate the biotin labeling efficiency of in vitro or in vivo biotinylation procedures, a streptavidin pull-down assay was performed. ComPACT proteins (EBV or MART-1) were biotinylated by treating sorted media with BirA enzyme and the appropriate reaction components (enzymatic, in vitro) or by co-expressing ComPACT with BirA (in vivo), and then incubated with increasing concentrations of streptavidin-coated beads. Streptavidin-coated magnetic beads (Dynabeads, Thermo Fisher Scientific) were added to 20 μg of comPACT protein sample and incubated for 30 minutes at room temperature. The magnetic beads were separated at the bottom of the tube or well by magnetization, and the protein content of the supernatant was assayed by SDS-PAGE. Figure 25 shows the results of the SDS-PAGE gel. Near-complete biotinylation can be achieved by adding BirA enzyme during purification (in vitro) or by co-expressing BirA enzyme and comPACT protein (in vivo). Samples lacking streptavidin beads had significant amounts of biotinylated protein in the supernatant, whereas addition of increasing amounts of streptavidin beads removed almost all protein from the supernatant, indicating biotinylation of comPACT protein.

[0186] Example 7: Antigen-specific T cell staining and affinity assessment using comPACT protein To compare antigen-specific T cell staining using comPACT with conventional peptide-MHC, comPACT dextramers were prepared according to a published protocol (Bethune, MT et al., BioTechniques 62, 123–130, doi:10.2144 / 000114525(2017)). T cells were engineered to express an A2 / neo12-specific TCR and stained with either HLA-A2 / neo12 peptide-MHC dextramers or HLA-A2 / neo12 peptide comPACT dextramers. Staining with comPACT dextramers was at least as efficient as peptide-MHC dextramers (Figure 19). This data suggests that comPACT dextramers can be used to sort antigen-specific T cells for TCR sequencing.

[0187] Example 8: Functional T cell assay NTAmer binding assay material and method Beyond antigen-specific capture of T cells, comPACT's modular design and easy generation facilitate its use in functional T cell assays. For example, incorporation of a mutant version of β2M (S88C) allows comPACT to be labeled with a maleimide-dye conjugate, assembled as an NTAmer, and used to measure the kinetic parameters of TCR-comPACT binding. For example, NTAmer can be used to resolve monovalent TCR-MHC I binding events in live cells. CD8 binding and multiple TCR-MHC I interactions at the immune synapse allow for prolonged contact between T cells and antigen-presenting cells. A fluorescent dye-conjugated ComPACT NTAmer is incubated with cells to allow binding of comPACT to the TCR. The Ni-PE particle component of the NTAmer is dissociated from comPACT by the addition of imidazole, and the release of fluorescent comPACT is measured over time.

[0188] Dye conjugation The S88C mutant comPACT protein was constructed and expressed at approximately 150 mg / L. These mutant comPACTs exhibited similar purity and elution profiles to unmutated comPACT (Figure 20). Other dyes, such as Cy5, can also be conjugated to S88C comPACT. The comPACT88C-HisTag monomer was reduced with Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and coupled with maleimide-Cy5 to yield the comPACT88c-Cy5-labeled monomer. Figure 21 shows quantification of the A280, A260, and A650 values ​​of Cy5-labeled comPACT. The dye-to-comPACT ratio was 67%.

[0189] NTAmer assembly Biotinylated comPACT was conjugated to PE-biotin-NTA beads by adding nickel (Ni) to biotin-NTA (biotin-NiNTA) and then assembling comPACT-Cy5 with biotin-NiNTA and PE-streptavidin (SA). Biotin-NiNTA was prepared by mixing 5 mg of biotin-NTA with 1 mL of nickel loading solution (50 mM NiSO4 in 100 mM HEPES pH 7.5). 2+ NTA-biotin (7 nM) was obtained. The solution was diluted with HBS to give 70 μM Ni 2+ An NTA-biotin solution was obtained. 2+The NTA-biotin solution was combined with SA-PE (300 kDa) in five additions, with 5 minutes between each addition. Next, 20 μM Cy5-S88C comPACT was added to the NTAmer core, incubated for 10 minutes at room temperature in the dark, and stored at 4°C. Both Neo12 and MART1 comPACT were generated and assembled into NTAmers. The His tag on the comPACT protein binds to NiNTA, which itself binds to PE-streptavidin. Each NTAmer contains multiple copies of the comPACT monomer bound to a streptavidin core bearing a fluorophore. The assembly of NTAmers is further discussed in Schmidt et al., J. Biol Chem, December 2, 2011, Vol. 286(48), pp. 41723-41735, and Schmidt et al., Front Immunol, July 30, 2013, doi:10.3389 / fimmu.2013.00218, both of which are incorporated by reference in their entirety.

[0190] Biotinylated Neo12 and MART1 comPACT proteins were also assembled into PE-labeled tetramers and dextramers. Peptide-linked refolded MHC I molecules, along with neo12 antigen peptides, were also assembled into PE-labeled dextramers as controls. Methods for assembling peptide-MHC proteins into tetramers and dextramers are generally known in the art.

[0191] T cell binding 1–2 × 10 gene-edited T cells expressing TCRs that bind to Neo12 or MART1 neoantigens 6Cells were resuspended in 1x staining buffer (BD Bovine Serum Albumin Staining Buffer, BD554657) at a density of 1000 cells / mL. Cells were maintained at 4°C throughout the assay. NTAmers were added to T cells in staining buffer at various concentrations, ranging from 1:50 to 1:400 (v:v dilution), and incubated for 15 minutes at 4°C in the dark to allow multimerized NTAmers to bind to T cells. T cells were washed twice with 1x staining buffer to remove unbound NTAmers and resuspended in staining buffer. To disrupt multimerized NTAmers and monitor Cy5 signal decay, imidazole disruption buffer (1.452 g imidazole and 50 mL 1x Hank's buffered saline) was added, mixed, and the samples were evaluated via flow cytometer to monitor Cy5 signal decay. Further antibody staining for the detection of various cell markers was performed by incubating T cells for 15 minutes at 4°C in the dark and washing the cells twice in 1x staining buffer. Next, T cells were fixed using IC fixation buffer (eBioscience IC fixation buffer, 00-8222-49).

[0192] result First, we assessed the ability of biotinylated comPACT-Cy5 NTAmer to bind to the cognate TCR. Neo12 peptide-linked refolded MHC I molecules, neo12 comPACT molecules, or neo12 NTAmer were assembled into dextramers and incubated with T cells expressing the neo12 TCR. The binding of each dextramer molecule to the cells was determined. Figure 28A shows that gene-edited T cells expressing the neo12 TCR exhibited specific binding to the neo12 peptide-linked refolded MHC I molecule dextramer (lower plot, x-axis is dextramer PE signal), which corresponds to a T cell population expressing CD3 on their surface (upper plot, x-axis is PE-Cy3 signal). Figure 28B shows that gene-edited T cells expressing the neo12 TCR show specific binding to the neo12 comPACT molecule dextramer (lower plot, x-axis is dextramer PE signal), which corresponds to a T cell population expressing CD3 on their surface (upper plot, x-axis is PE-Cy3 signal). Figure 28C shows that gene-edited T cells expressing the neo12 TCR show specific binding to the neo12 comPACT NTAmer dextramer (lower plot, x-axis is dextramer PE signal), which corresponds to a T cell population expressing CD3 on their surface (upper plot, x-axis is PE-Cy3 signal).

[0193] Next, we confirmed the ability of the biotinylated comPACT-Cy5 NTAmer to bind antigen-specifically to TCRs. Neo12 or F5 TCR T cells were incubated with the NTAmer and neo12 antigen. F5 TCR T cells were used as a negative control. Neo12 TCR T cells were also incubated with a dextramer composed of the neo12 comPACT protein. Bound T cells were then incubated with imidazole as described, and binding of the NTAmer and dextramer was assessed as previously described. The addition of imidazole inhibited the ability of the neo12-ComPACT NTAmer to bind to neo12 T cells (Figure 29A-C). Neo12 TCR T cells showed specific binding to the neo12 NTAmer reagent (Figure 29A, NTAmer Cy5 signal is on the y-axis). The neo12-ComPACT NTAmer was specific for Neo12 T cells and did not bind to T cells expressing the F5 TCR (Figure 29B). The dextramer of Neo12 comPACT also bound to neo12 TCR T cells (Figure 29D). Neo12 TCR T cells showed no binding to the neo12 NTAmer reagent when incubated in the presence of imidazole (Figure 29C). This demonstrated the need for a multimerization reagent to observe Cy5 signal from S88C-modified comPACT molecules.

[0194] We also used MART-1 NTAmer to demonstrate antigen-specific binding of T cells edited to express the F5 TCR (Figures 30A-D). Figure 30A shows the binding of MART-1 comPACT dextramers to F5 TCR T cells. Figure 30B shows the binding of MART-1 comPACT dextramers to M1W TCR T cells. Figure 30C shows the binding of MART-1 comPACT NTAmers to F5 TCR T cells. Figure 30D shows the binding of MART-1 comPACT NTAmers to M1W TCR T cells. The low level of binding of MART-1 comPACT NTAmers to M1W TCR T cells may be due to the low affinity of the M1W TCR for its cognate antigen:MHC I protein.

[0195] Dissociation of monomeric TCR:pMHC-Cy5 was assessed by measuring Cy5 signal decay of two TCRs, MART-1 F5 and M1W, with different TCR:MHC I binding affinities for the same peptide:MHC I complex (HLA:A02 + MART-1). Binding of the MART1 NTAmer to T cells expressing the F5 TCR was stable over time in the absence of imidazole, which induces NTAmer disruption (Figure 31A). However, disruption of the multimerized MART-1 NTAmer with imidazole resulted in a time-dependent dissociation of monomeric MART-1 comPACT molecules from the F5 TCR (Figure 31B). Figure 31C shows a visually expanded subset of the time data from Figure 31B.

[0196] In conclusion, Neo12 and F5 T cells exhibit similar binding levels between the NTAmer and the equivalent comPACT dextramer, demonstrating that the comPACT NTAmer can bind to neoantigen-specific T cells. Neo12 and F5 T cells demonstrate similar binding levels between the NTAmer and comPACT dextramer for both neo12 and MART-1 comPACT. Furthermore, the NTAmer complex can be disrupted by the addition of imidazole, and the rate of monovalent TCR:MHC I dissociation can be measured by the decay of the comPACT-Cy5 signal on live T cells. This decay occurs on a time scale of tens of seconds.

[0197] Example 9: comPACT library generation The diversity of HLA alleles in the US human population was analyzed bioinformatically from the Allele Frequency Net database (www.allelefrequencies.net) to identify the optimal number of alleles to include in the HLA repertoire that would result in high coverage of the target HLA frequency. 9,736 alleles were analyzed. Figure 26A shows an analysis of the percentage of patients in which one or both alleles from each of the HLA A, B, and C loci are covered by a library of 66 HLA alleles. Solid lines indicate coverage of one allele, and dotted lines indicate coverage of both alleles. The 66 alleles allow coverage of at least four of the six HLA alleles per patient in over 95% of the total population, and coverage of six of the six alleles in over 80% of the population (Figure 26B). The most frequent HLA-I allele is HLA-A02:01, with a prevalence of approximately 50% in the US. Thus, the HLA library presented here for the first time is global and enables the greatest potential for widespread implementation of personalized neoTCR-T cell therapy for diverse populations.

[0198] Next, a library of comPACT proteins bearing different neoepitopes and selected HLA alleles was generated. Neoepitope candidates were selected from the Immune Epitope Database (www.iedb.org). Complete sequences for each of the 66 HLA-I alleles in the repertoire were obtained from the IMGT database and modified to include the Y84C mutation. All clones were sequence-verified and stored in the database and reagent inventory. Ten neoepitope peptides were selected from the IEDB database and inserted into a panel of 36 HLA alleles. ComPACT proteins bearing the selected neoepitopes and HLA alleles were expressed and purified via a size-exclusion chromatography column (Agilent Sec Bio 300) connected to an Agilent Infinity II HPLC system (SEC-HPLC) according to the manufacturer's instructions. The results are shown in Figures 27A-C. comPACTs were purified as monodisperse polypeptides and assessed by SEC-HPLC by measuring the area under the curve of the monomer peak divided by the area under the total chromatogram (Figures 27A and 27B). Most comPACTs were expressed at high titers (Figure 27C). At least one comPACT protein was purified and characterized via HPLC for each listed HLA allele, demonstrating that the comPACT platform is robust and sensitive to many alleles.

[0199] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

[0200] All references, issued patents and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes. TIFF2024170434000005.tif221170TIFF2024170434000006.tif219170TIFF2024170434000007.tif221170TIFF2024170434000008.tif217170TIFF2024170434000009.tif220170TIFF2024170434000010.tif214170TIFF2024170434000011.tif217170TIFF2024170434000012.tif220170TIFF2024170434000013.tif221170TIFF2024170434000014.tif219170TIFF2024170434000015.tif220170TIFF2024170434000016.tif223170TIFF2024170434000017.tif221170TIFF2024170434000018.tif221170TIFF2024170434000019.tif219170TIFF2024170434000020.tif221170TIFF2024170434000021.tif220170TIFF2024170434000022.tif222170TIFF2024170434000023.tif217170TIFF2024170434000024.tif222170TIFF2024170434000025.tif220170TIFF2024170434000026.tif222170TIFF2024170434000027.tif210170(Sequence Listing) SEQUENCE LISTING <110> PACT PHARMA, INC. <120> PEPTIDE-MHC COMPACTS <130> 087520.0114 <150> PCT / US2019 / 025415 <151> 2019-04-02 <150> 62 / 651,639 <151> 2018-04-02 <160> 218 <170> PatentIn version 3.5 <210> 1 <211> 78 <212> DNA <213> Homo sapiens <400> 1 atggcgacgg gttcaagaac ttccctactt cttgcatttg gcctgctttg tttgccgtgg 60 ttacaggagg gctcagca 78 <210> 2 <211> 26 <212> PRT <213> Homo sapiens <400> 2 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala 20 25 <210> 3 <211> 23 <212> DNA <213> Unknown <220> <223> Description of Unknown: Universal target sequence <400> 3 cgtggttaca ggagggctca gca 23 <210> 4 <211> 19 <212> DNA <213> Unknown <220> <223> Description of Unknown: Universal target sequence <400> 4 ggatgcggag gatccggcg 19 <210> 5 <211> 19 <212> DNA <213> Unknown <220> <223> Description of Unknown: Universal target sequence <400> 5 ggaagcggag gatccggcg 19 <210> 6 <211> 21 <212> DNA <213> Unknown <220> <223> Description of Unknown: Universal target sequence <400> 6 ggaagcggag gatccaccag c 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 atgtacgggc cagatatacg c 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 acacccgccg cgcttaatg 19 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Gly Gly Gly Gly Ser 1 5 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 ggcggcggcg gcagc 15 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Gly Ser Gly Gly Ser 1 5 <210> 12 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 12 ggcagcggcg gcagc 15 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Gly Cys Gly Gly Ser 1 5 <210> 14 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 14 ggctgcggcg gcagc 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Gly Cys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 16 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 ggctgcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 18 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45 <210> 19 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 20 ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 60 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Gly Ser Gly Gly Ser Gly Gly Ser Ala Gly Gly 1 5 10 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 ggcagcggcg gcagcggcgg cagcgcgggc ggc 33 <210> 23 <211> 22 <212> PRT <213> Homo sapiens <400> 23 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys 20 <210> 24 <211> 66 <212> DNA <213> Homo sapiens <400> 24 atggatatgc gcgtgccggc gcagctgctg ggcctgctgc tgctgtggct gagcggcgcg 60 cgctgc 66 <210> 25 <211> 20 <212> PRT <213> Unknown <220> <223> Description of Unknown: Beta 2 microglobulin sequence <400> 25 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala 20 <210> 26 <211> 60 <212> DNA <213> Unknown <220> <223> Description of Unknown: Beta 2 microglobulin sequence <400> 26 atgagccgca gcgtggcgct ggcggtgctg gcgctgctga gcctgagcgg cctggaagcg 60 <210> 27 <211> 20 <212> PRT <213> Unknown <220> <223> Description of Unknown: IL2 signal sequence <400> 27 Put Tyr Arg Put Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 <210> 28 <211> 60 <212> DNA <213> Unknown <220> <223> Description of Unknown: IL2 signal sequence <400> 28 atgtatcgca tgcagctgct gagctgcatt gcgctgagcc tggcgctggt gaccaacagc 60 <210> 29 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 29 ggcctgaacg acatcttcga ggctcagaaa atcgaatggc acgaa 45 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 31 <211> 23 <212> DNA <213> Unknown <220> <223> Description of Unknown: TEV cleavage site <400> 31 gagagaacct gtacttccag ggc 23 <210> 32 <211> 7 <212> PRT <213> Unknown <220> <223> Description of Unknown: TEV cleavage site <400> 32 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 33 catcatcatc atcatcat 18 <210> 34 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 34 His His His His His His 1 5 <210> 35 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 35 catcatcatc atcatcatgg cggcggcagc ggcggcggca gcggcagcca tcatcatcat 60 catcat 66 <210> 36 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 His His His His His His Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser 1 5 10 15 His His His His His His 20 <210> 37 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly 1 5 10 15 Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly Ser His His His His His 20 25 30 His Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser His His His His His 35 40 45 His <210> 38 <211> 576 <212> DNA <213> Cytomegalovirus <400> 38 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 60 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 120 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 180 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 240 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 300 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 360 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 420 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 480 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 540 gtctatataa gcagagctgg tttagtgaac cgtcag 576 <210> 39 <211> 276 <212> PRT <213> Homo sapiens <400> 39 Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Lys Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Met Lys Ala His Ser Gln Thr Asp Arg Ala Asn Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile Gln 85 90 95 Ile Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Lys 130 135 140 Arg Lys Trp Glu Ala Val His Ala Ala Glu Gln Arg Arg Val Tyr Leu 145 150 155 160 Glu Gly Arg Cys Val Asp Gly Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Leu 275 <210> 40 <211> 276 <212> PRT <213> Homo sapiens <400> 40 Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr 50 55 60 Arg Lys Val Lys Ala His Ser Gln Thr His Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Val Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp Arg Phe Leu Arg Gly 100 105 110 Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys 130 135 140 His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 41 <211> 276 <212> PRT <213> Homo sapiens <400> 41 Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Ile Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Lys 130 135 140 Arg Lys Trp Glu Ala Ala His Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Asp Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Leu 275 <210> 42 <211> 276 <212> PRT <213> Homo sapiens <400> 42 Gly Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Glu Glu Thr 50 55 60 Gly Lys Val Lys Ala His Ser Gln Thr Asp Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Met Met Phe Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Lys 130 135 140 Arg Lys Trp Glu Ala Ala His Val Ala Glu Gln Gln Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Asp Gly Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 43 <211> 276 <212> PRT <213> Homo sapiens <400> 43 Gly Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Ser Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Arg Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Thr Asp Arg Glu Asn Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Ile Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Glu Gln His Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Arg Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Leu 275 <210> 44 <211> 276 <212> PRT <213> Homo sapiens <400> 44 Gly Ser His Ser Met Arg Tyr Phe Thr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Arg Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Ile Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Met Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Gln Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 45 <211> 276 <212> PRT <213> Homo sapiens <400> 45 Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Thr Asp Arg Glu Ser Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Met Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Gln Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 46 <211> 276 <212> PRT <213> Homo sapiens <400> 46 Gly Ser His Ser Met Arg Tyr Phe Thr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Ile Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Met Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Gln Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg His Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Arg Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 47 <211> 276 <212> PRT <213> Homo sapiens <400> 47 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Met Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys 130 135 140 His Lys Trp Glu Ala Ala His Val Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Val Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 48 <211> 276 <212> PRT <213> Homo sapiens <400> 48 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Tyr Lys Ala Gln Ala Gln Thr Asp Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Asp Lys Leu Glu Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 49 <211> 276 <212> PRT <213> Homo sapiens <400> 49 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Cys Lys Thr Asn Thr Gln Thr Asp Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Trp Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg His Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 50 <211> 276 <212> PRT <213> Homo sapiens <400> 50 Gly Ser His Ser Met Arg Tyr Phe His Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Gly Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg His Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 51 <211> 276 <212> PRT <213> Homo sapiens <400> 51 Gly Ser His Ser Met Arg Tyr Phe His Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Cys Lys Ala Lys Ala Gln Thr Asp Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Asn Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr His Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 52 <211> 276 <212> PRT <213> Homo sapiens <400> 52 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Cys Lys Thr Asn Thr Gln Thr Asp Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Thr Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 53 <211> 276 <212> PRT <213> Homo sapiens <400> 53 Gly Ser His Ser Met Arg Tyr Phe His Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Gln 130 135 140 Arg Lys Leu Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Asp Lys Leu Glu Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 54 <211> 276 <212> PRT <213> Homo sapiens <400> 54 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Thr 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Ser Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Val Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 55 <211> 276 <212> PRT <213> Homo sapiens <400> 55 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Ala Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 56 <211> 276 <212> PRT <213> Homo sapiens <400> 56 Gly Ser His Ser Met Arg Tyr Phe His Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Trp Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Leu Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 57 <211> 276 <212> PRT <213> Homo sapiens <400> 57 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Ala Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr 50 55 60 Arg Asn Met Lys Ala Ser Ala Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Val Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 58 <211> 276 <212> PRT <213> Homo sapiens <400> 58 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr 50 55 60 Arg Asn Met Lys Ala Ser Ala Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 59 <211> 276 <212> PRT <213> Homo sapiens <400> 59 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Ser 1 5 10 15 Arg Gly Glu Pro His Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Asn Leu Arg Lys 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Thr Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 60 <211> 276 <212> PRT <213> Homo sapiens <400> 60 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro His Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Trp Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 61 <211> 276 <212> PRT <213> Homo sapiens <400> 61 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Gln Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Asn Leu Arg Lys 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Lys Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Lys Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Gly Pro 275 <210> 62 <211> 276 <212> PRT <213> Homo sapiens <400> 62 Cys Ser His Ser Met Arg Tyr Phe Asp Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Asn Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Leu Glu Ala Ala Arg Ala Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Leu Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Met Gln His Glu Gly Leu Gln Glu Pro Leu Thr Leu 260 265 270 Ser Trp Glu Pro 275 <210> 63 <211> 276 <212> PRT <213> Homo sapiens <400> 63 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile Gln 85 90 95 Ile Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Lys 130 135 140 Arg Lys Trp Glu Ala Ala His Ala Ala Glu Gln Gln Arg Ala Tyr Leu 145 150 155 160 Glu Gly Arg Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Leu 275 <210> 64 <211> 276 <212> PRT <213> Homo sapiens <400> 64 Gly Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Glu Glu Thr 50 55 60 Gly Lys Val Lys Ala His Ser Gln Thr Asp Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Met Met Phe Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Asp Gly Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 65 <211> 276 <212> PRT <213> Homo sapiens <400> 65 Gly Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Ser Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Arg Pro Glu Tyr Trp Asp Gln Glu Thr 50 55 60 Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Ile Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Glu Gln His Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Trp Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Leu 275 <210> 66 <211> 276 <212> PRT <213> Homo sapiens <400> 66 Gly Ser His Ser Met Arg Tyr Phe Thr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Ile Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Met Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Gln Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Pro Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 67 <211> 276 <212> PRT <213> Homo sapiens <400> 67 Gly Ser His Ser Met Arg Tyr Phe Asp Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys Thr Asn Thr Gln Thr Asp Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Asp Thr Leu Glu Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 68 <211> 276 <212> PRT <213> Homo sapiens <400> 68 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Ala Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 69 <211> 276 <212> PRT <213> Homo sapiens <400> 69 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 70 <211> 276 <212> PRT <213> Homo sapiens <400> 70 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 71 <211> 276 <212> PRT <213> Homo sapiens <400> 71 Gly Ser His Ser Met Arg Tyr Phe His Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 72 <211> 276 <212> PRT <213> Homo sapiens <400> 72 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Tyr Lys Ala Gln Ala Gln Thr Asp Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Asp Thr Leu Glu Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 73 <211> 276 <212> PRT <213> Homo sapiens <400> 73 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Thr 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Ser Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Val Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 74 <211> 276 <212> PRT <213> Homo sapiens <400> 74 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Trp Gln 85 90 95 Thr Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg His Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 75 <211> 276 <212> PRT <213> Homo sapiens <400> 75 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 76 <211> 276 <212> PRT <213> Homo sapiens <400> 76 Cys Ser His Ser Met Lys Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Trp Met Cys Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Trp Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Met Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 77 <211> 276 <212> PRT <213> Homo sapiens <400> 77 Gly Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Trp Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Glu Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Asn Leu Arg Lys 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Leu Gln 85 90 95 Arg Met Phe Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Trp Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Lys Pro 275 <210> 78 <211> 275 <212> PRT <213> Homo sapiens <400> 78 Cys Ser His Ser Met Arg Tyr Phe Asp Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Asn Leu Arg Lys 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Leu Gln 85 90 95 Trp Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu 275 <210> 79 <211> 276 <212> PRT <213> Homo sapiens <400> 79 Cys Ser His Ser Met Arg Tyr Phe Asp Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Leu Gln 85 90 95 Arg Met Ser Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Leu Glu Ala Ala Arg Ala Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Leu Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Met Gln His Glu Gly Leu Gln Glu Pro Leu Thr Leu 260 265 270 Ser Trp Glu Pro 275 <210> 80 <211> 276 <212> PRT <213> Homo sapiens <400> 80 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Trp Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Ala Ala Glu Gln Gln Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Leu Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 81 <211> 276 <212> PRT <213> Homo sapiens <400> 81 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Thr Asp Arg Glu Ser Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Gln Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Thr Ala His Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Arg Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 82 <211> 276 <212> PRT <213> Homo sapiens <400> 82 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Arg Asn Val Lys Ala His Ser Gln Thr Asp Arg Ala Asn Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Gln Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Thr Ala His Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Arg Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 83 <211> 276 <212> PRT <213> Homo sapiens <400> 83 Gly Ser His Ser Met Arg Tyr Phe Thr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Leu Gln Thr 50 55 60 Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Ala Asn Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Met Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 84 <211> 276 <212> PRT <213> Homo sapiens <400> 84 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Arg Asn Val Lys Ala Gln Ser Gln Thr Asp Arg Val Asp Leu Gly Thr 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg Gly 100 105 110 Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys 130 135 140 His Lys Trp Glu Ala Ala His Val Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Val Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 85 <211> 276 <212> PRT <213> Homo sapiens <400> 85 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Met Ala Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Thr 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Trp Gln 85 90 95 Thr Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Leu Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Leu Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 86 <211> 276 <212> PRT <213> Homo sapiens <400> 86 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Ala Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 87 <211> 276 <212> PRT <213> Homo sapiens <400> 87 Gly Ser His Ser Met Arg Tyr Phe His Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Cys Lys Ala Lys Ala Gln Thr Asp Arg Glu Asp Leu Arg Thr 65 70 75 80 Leu Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Asn Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr His Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 88 <211> 276 <212> PRT <213> Homo sapiens <400> 88 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 89 <211> 276 <212> PRT <213> Homo sapiens <400> 89 Gly Ser His Ser Met Arg Tyr Phe His Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asp Leu Arg Thr 65 70 75 80 Leu Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Arg Met Ser Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 90 <211> 276 <212> PRT <213> Homo sapiens <400> 90 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Cys Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg Thr Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 91 <211> 276 <212> PRT <213> Homo sapiens <400> 91 Gly Ser His Ser Met Arg Tyr Phe His Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Ser Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Asp Thr Leu Glu Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 92 <211> 276 <212> PRT <213> Homo sapiens <400> 92 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Thr 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Ser Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Val Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 93 <211> 276 <212> PRT <213> Homo sapiens <400> 93 Gly Ser His Ser Met Arg Tyr Phe His Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Thr Val Gly Tyr Val Asp Asp Thr Leu 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Thr Ser Pro Arg Lys Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Trp Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Leu Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 94 <211> 276 <212> PRT <213> Homo sapiens <400> 94 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Trp Gln 85 90 95 Thr Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg His Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 95 <211> 276 <212> PRT <213> Homo sapiens <400> 95 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Tyr Lys Ala Gln Ala Gln Thr Asp Arg Glu Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Trp Gln 85 90 95 Thr Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asn Gln Leu Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 96 <211> 276 <212> PRT <213> Homo sapiens <400> 96 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro His Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Arg Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Trp Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 97 <211> 276 <212> PRT <213> Homo sapiens <400> 97 Cys Ser His Ser Met Arg Tyr Phe Asp Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Phe Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Leu Glu Ala Ala Arg Ala Ala Glu Gln Asp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Lys Thr Leu Gln Arg Ala Glu Pro Pro Lys Thr His Val Thr His His 180 185 190 Pro Leu Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Met Gln His Glu Gly Leu Gln Glu Pro Leu Thr Leu 260 265 270 Ser Trp Glu Pro 275 <210> 98 <211> 276 <212> PRT <213> Homo sapiens <400> 98 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Gln Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Thr Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Lys Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Gly Pro 275 <210> 99 <211> 276 <212> PRT <213> Homo sapiens <400> 99 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Gln Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Lys Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Lys Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Gly Pro 275 <210> 100 <211> 276 <212> PRT <213> Homo sapiens <400> 100 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 101 <211> 276 <212> PRT <213> Homo sapiens <400> 101 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Trp Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Trp Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 102 <211> 276 <212> PRT <213> Homo sapiens <400> 102 Cys Ser His Ser Met Arg Tyr Phe Ser Thr Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Ser Leu Arg Asn 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95 Trp Met Phe Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Arg Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Trp Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 103 <211> 276 <212> PRT <213> Homo sapiens <400> 103 Cys Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro His Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Asn Tyr Lys Arg Gln Ala Gln Thr Asp Arg Val Asn Leu Arg Lys 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 His Asp Gln Leu Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu His Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro 275 <210> 104 <211> 276 <212> PRT <213> Homo sapiens <400> 104 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Gly Glu Pro Arg 35 40 45 Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Arg Glu Thr 50 55 60 Gln Lys Tyr Lys Arg Gln Ala Gln Ala Asp Arg Val Asn Leu Arg Lys 65 70 75 80 Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Ile Gln 85 90 95 Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100 105 110 Tyr Asn Gln Phe Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Gln 130 135 140 Arg Lys Leu Glu Ala Ala Arg Glu Ala Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Glu Cys Val Glu Trp Leu Arg Gly Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala Glu Arg Pro Lys Thr His Val Thr His His 180 185 190 Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Gln Glu Pro Cys Thr Leu 260 265 270 Arg Trp Lys Pro 275 <210> 105 <211> 99 <212> PRT <213> Unknown <220> <223> Description of Unknown: Beta 2 microglobulin sequence <400> 105 Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu 1 5 10 15 Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro 20 25 30 Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys 35 40 45 Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60 Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys 65 70 75 80 Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp 85 90 95 Arg Asp Met <210> 106 <211> 297 <212> DNA <213> Unknown <220> <223> Description of Unknown: Beta 2 microglobulin sequence <400> 106 atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 60 aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 120 aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 180 tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 240 cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatg 297 <210> 107 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 107 Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu 1 5 10 15 Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro 20 25 30 Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys 35 40 45 Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60 Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys 65 70 75 80 Arg Val Asn His Val Thr Leu Cys Gln Pro Lys Ile Val Lys Trp Asp 85 90 95 Arg Asp Met <210> 108 <211> 297 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 108 atccagcgta ctccaagat tcaggtttac tcacgtcatc cagcagagaa tggaagtca 60 aatttcctga attgctatgt gtctggtttt catccatccg acattgaagt tgacttactg 120 aagaatggag agagaattga aaagtggag cattcagact tgtctttcag caggactgg 180 tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 240 cgtgtgaacc atgtgactt gtgccagccc agatagtta agtgggatcg agacatg 297 <210> 109 <211> 828 <212> DNA <213> Homo sapiens <400> 109 ggctcccact ccatgaggta tttctcaca tccgtgtccc ggcccggccg cggggagcccc 60 cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga agatggagcc gcggggccg tggatagagc aggaggggcc ggagtattgg 180 gaccaggaga cacggaatat gaaggcccac tcacagactg accgagcgaa cctggggacc 240 ctgcgcggct gctacaacca gagcgaggac ggttctcaca ccatccagat aatgtatggc 300 tgcgacgtgg ggccggacgg gcgcttcctc cgcgggtacc ggcaggacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcttgga ccgcggcgga catggcagct 420 cagatcacca agcgcaagtg ggaggcggtc catgcggcgg agcagcggag agtctacctg 480 gagggccggt gcgtggacgg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcggctgt ggtggtgcct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagctg 828 <210> 110 <211> 828 <212> DNA <213> Homo sapiens <400> 110 ggctctcact ccatgaggta tttcttcaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggagggtcc cgagtattgg 180 240. 240. 240. 240. 240. 240. 240. 240. 240. 240. 240 ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccgtccagag gatgtatggc tgcgacgtgg ggtcggactg gcgcttcctc cgcgggtacc accagtacgc ctacgacggc 360 aaggattaca tcgccctgaa agaggacctg cgctcttgga ccgcggcgga catggcagct cagaccacca agcacaagtg ggaggcggcc catgtggcgg agcagttgag agcctacctg 480. gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacg cccccaaaac gcatatgact caccacgctg tctctgacca tgaagccacc ctgaggtgct gggccctgag cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc ttccagaagt gggcggctgt ggtggtgcct tctggacagg agcagagata cacctgccat 780 gtgcagcatg agggtttgcc caagcccctc accctgagat gggagccg 828 <210> 111 <211> 828 <212> DNA <213> Homo sapiens <400> 111 60. ggctcccact ccatgaggta tttcttcaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccaggaga cacggaatgt gaaggcccag tcacagactg accgagtgga cctggggacc 240 ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat aatgtatggc 300 tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtacc ggcaggacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcttgga ccgcggcgga catggcggct 420 cagatcacca agcgcaagtg ggaggcggcc catgaggcgg agcagttgag agcctacctg 480 gatggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcggctgt ggtggtgcct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagctg 828 <210> 112 <211> 828 <212> DNA <213> Homo sapiens <400> 112 60. ggctcccact ccatgaggta tttctccaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gacgaggaga gaggaagt gaggcccac tcacagactg accgagaga cctgcggatc 300. gcgctccgct gctacaacca gagcgaggcc ggttctcaca ccctccagat gatgtttggc tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtacc accagtacgc ctacgacggc 360 aaggattaca tcgccctgaa agaggacctg cgctcttgga ccgcggcgga catggcggct cagatcacca agcgcaagtg ggaggcggcc catgtggcgg agcagcag agcctacctg 480 gagggcacgt gcgtggacgg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccact 600 ctgagatgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctt gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcagctgt ggtggtacct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagcca 828 <210> 113 <211> 828 <212> DNA <213> Homo sapiens <400> 113 ggctcccact ccatgaggta tttctccaca tccgtgtccc ggcccggcag tggagagccc 60 cgcttcatcg cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcgggcgccg tggatagagc aggagaggcc tgagtattgg 180 gaccaggaga cacggaatgt gaaggcccac tcacagactg accgagagaa cctggggacc 240 ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat aatgtatggc 300 tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtatg aacagcacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcttgga ccgcggcgga catggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtcgggcgg agcagttgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcggctgt ggtggtgcct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagctg 828 <210> 114 <211> 828 <212> DNA <213> Homo sapiens <400> 114 60. ggctcccact ccatgaggta tttcaccaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggagaggcc tgagtattgg 180 gaccaggaga cacggaatgt gaaggcccac tcacagattg accgagtgga cctggggacc ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat gatgtatggc tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtacc agcaggacgc ctacgacggc 360 aaggattaca tcgccttga cgaggacctg cgctcttgga ccgcggcgga catggcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagttgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac gcatatgact caccacgctg tctctgacca tgaggccacc 600 ctgaggtgct gggccctgag cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc ttccagaagt gggcgtctgt ggtggtgcct tctggacagg agcagagata cacctgccat 780 gtgcagcatg agggtctccc caagcccctc accctgagat gggagccg 828 <210> 115 <211> 828 <212> DNA <213> Homo sapiens <400> 115 60. ggctcccact ccatgaggta tttcttcaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggtttga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccaggaga cacggaatgt gaaggcccac tcacagactg accgagagag cctgcggatc 240 gcgctccgct gctacaacca gagcgaggcc ggttctcaca ccatccagat gatgtatggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggtacc agcaggacgc ctacgacggc 360 aaggattaca tcgccttgaa cgaggacctg cgctcttgga ccgcggcgga catggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagttgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacg cccccaagac gcatatgact caccacgctg tctctgacca tgaggccacc 600 ctgaggtgct gggccctgag cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctt gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcgtctgt ggtggtgcct tctggacagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagccg 828 <210> 116 <211> 828 <212> DNA <213> Homo sapiens <400> 116 60. ggctcccact ccatgaggta tttcaccaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccggaca cacggatgt gaggcccac tcacagattg accgagtgga cctggggacc ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat gatgtatggc tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtacc agcaggacgc ctacgacggc 360 aaggattaca tcgccttga cgaggacctg cgctcttgga ccgcggcgga catggcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagttgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga cacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaggac gcatatgact caccacgctg tctctgacca tgaggccacc 600 ctgaggtgct gggccctgag cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc ttccagaagt gggcgtctgt ggtggtgcct tctggacagg agcagagata cacctgccat 780 gtgcagcatg agggtctccc caagcccctc accctgagat gggagccg 828 <210> 117 <211> 828 <212> DNA <213> Homo sapiens <400> 117 60. ggctcccact ccatgaggta tttctacacc tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccggaaca cacggaatgt gaaggcccag tcacagactg accgagtgga cctgggggacc 240 ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat gatgtatggc 300 tgcgacgtgg ggtcggacgg gcgcttctcc cgcgggtacc ggcaggacgc ctacgacggc 360 aaggattaca tcgccctgaa agaggacctg cgctcttgga ccgcggcgga catggcagct 420 cagaccacca agcacaagtg ggaggcggcc catgtggcgg agcagtggag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacg cccccaaaac gcatatgact caccacgctg tctctgacca tgaagccacc 600 ctgaggtgct gggccctgag cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggtggctgt ggtggtgcct tctggacagg agcagagata cacctgccat 780 gtgcagcatg agggtttgcc caagcccctc accctgagat gggagccg 828 <210> 118 <211> 828 <212> DNA <213> Homo sapiens <400> 118 60. ggctcccact ccatgaggta tttctacacc tccgtgtccc ggcccggccg cggggagccc cgcttcatct cagtgggcta cgtggacgac acccagttcg tgaggttcga cagcgacgcc 120 gcgagtccga gagaggagcc gcgggcgccg tggatagc aggaggggcc ggagtattgg 180 240. gaccggaca cacagatcta caaggcccag gcacagactg accgagagag cctgcggaac 300. ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag catgtacggc tgcgacgtgg ggccggacgg gcgcctcctc cgcgggcatg accagtacgc ctacgacggc 360 aaggattaca tcgccctga cgaggacctg cgctcctgga ccgccgcgga cacggcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagcggag agcctacctg 480 gagggcgagt gcgtggagtg gctccgcaga tacctggaga acgggaagga caagctggag 540 600. cgcgctgacc ccccaaagac acacgtgacc cccccc tctctgacc tgaggccacc ctgaggtgct gggccctggg tttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga cactgagctt gtggagacca gaccagcagg agatagaacc ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagccg 828 <210> 119 <211> 828 <212> DNA <213> Homo sapiens <400> 119 60. ggctcccact ccatgaggta tttctacacc gccgtgtccc ggcccggccg cggggagccc cgcttcatct cagtgggcta cgtggacgac acgcagttcg tgaggttcga cagcgacgcc 120 gcgagtccga gagaggagcc gcgggcgccg tggataggc aggaggggcc ggaatattgg gaccggaaca cacagatctg caagaccaac acacagactg accgagagag cctgcggaac 240 ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagtg gatgtatggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggtata accagttcgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg agctcctgga ccgcggcgga caccgcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagctgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga cacctggaga acgggaagga gacgctgcag 540 cgcgcggacc ccccaaagac acatgtgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agacagaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagcca 828 <210> 120 <211> 828 <212> DNA <213> Homo sapiens <400> 120 ggctccccact ccatgaggta tttccacacc tccgtgtccc ggcccggccg cggggagccc 60 cgcttcatct cagtgggcta cgtggacggc acccagttcg tgaggttcga cagcgacgcc 120 gcgagtccga ggacggagcc ccgggcgccg tggatagagc aagaggggcc ggagtattgg 180 gaccggaaca cacagatctc caagaccaac acacagactt accgagagag cctgcggaac 240 ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag gatgtacggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggcatg accagtccgc ctacgacggc 360 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagctgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga cacctggaga acgggaagga gacgctgcag 540 600. cgcgcggacc ccccaaagac acatgtgacc cccccc tctctgacc tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agatagaacc ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagcca 828 <210> 121 <211> 828 <212> DNA <213> Homo sapiens <400> 121 60. ggctcccact ccatgaggta tttccacacc tccgtgtccc ggcccggccg cggggagccc cgcttcatca ccgtgggcta cgtggacgac acgctgttcg tgaggttcga cagcgacgcc 120 gcgagtccga gagaggagcc gcgggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccgggaga cacagatctg caaggccaag gcacagactg accgagagaa cctgcggatc 240 gcgctccgct gctacaacca gagcgaggcc gggtctcaca ccctccagaa tatgtatggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggtacc accaggacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg agctcctgga ccgccgcgga cacggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagctgag agcctacctg 480 gagggcgagt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcgcggacc ccccaaagac acacgtgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga cactgagctt gtggagacca gaccagcagg agatagaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagccg 828 <210> 122 <211> 828 <212> DNA <213> Homo sapiens <400> 122 60. ggctcccact ccatgaggta tttctacacc tccgtgtccc ggcccggccg cggggagccc cgcttcatct cagtgggcta cgtggacgac acgcagttcg tgaggttcga cagcgacgcc 120 gcgagtccga gagaggagcc gcgggcgccg tggataggc aggaggggcc ggaatattgg 240. gaccggaca cacagatctg caagaccaac acacagactg accgagagag cctgcggaac ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag gatgtacggc tgcgacgtgg ggccggacgg gcgcctcctc cgcgggcata accagttcgc ctacgacggc 360 aaggattaca tcgccctga cgaggacctg agctcctgga ccgcggcgga caccgcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagctgag aacctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 600. cgcgcggacc ccccaaagac acatgtgacc cccccc tctctgacc tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agacagaacc ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagcca 828 <210> 123 <211> 828 <212> DNA <213> Homo sapiens <400> 123 60. ggctcccact ccatgaggta tttccacacc gccatgtccc ggcccggccg cggggagccc cgcttcatca ccgtgggcta cgtggacgac acgctgttcg tgaggttcga cagcgacgcc 120 acgagtccga ggaggagcc gcggggcgcca tggatagc aggaggggcc ggagtattgg gaccgggaga cacagatctc caagaccaac acacagactt accgagagag cctgcggaac 240 ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag gatgtacggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggcata accagtacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcctgga ccgccgcgga cacggcggct 420 cagatctccc agcgcaagtt ggaggcggcc cgtgtggcgg agcagctgag agcctacctg 480 gagggcgagt gcgtggagtg gctccgcaga tacctggaga acgggaagga caagctggag 540 cgcgctgacc ccccaaagac acacgtgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg tttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga cactgagctt gtggagacca gaccagcagg agatagaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagccg 828 <210> 124 <211> 828 <212> DNA <213> Homo sapiens <400> 124 60. ggctcccact ccatgaggta tttctacacc gccatgtccc ggcccggccg cggggagccc cgcttcatca ccgtgggcta cgtggacgac acgctgttcg tgaggttcga cagcgacgcc 120 acgagtccga ggaggagcc gcggggcgcca tggatagc aggaggggcc ggagtattgg gaccgggaga cacagatctc caagaccaac acacagactt accgagaga cctgcgcacc gcgctccgct gctacaacca gagcgaggcc gggtctcaca tcatccagag gatgtacggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggtatg accaggacgc ctacgacggc 360 aaggattaca tcgccctga cgaggacctg agctcctgga ccgcggcgga caccgcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagacag agcctacctg 480 gagggcctgt gcgtggagtc gctccgcaga tacctggaga acgggaagga gacgctgcag 540 600. cgcgcggacc ccccaaagac acatgtgacc cccccc tctctgacca tgaggtcacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agatagaacc ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagccg 828 <210> 125 <211> 828 <212> DNA <213> Homo sapiens <400> 125 60. ggctcccact ccatgaggta tttctacacc gccatgtccc ggcccggccg cggggagccc cgcttcatcg cagtgggcta cgtggacgac acccagttcg tgaggttcga cagcgacgcc 120 gcgagtccga ggatggcgcc ccggggcgcca tggatagc aggaggggcc ggagtattgg 180 gaccgggaga cacagaagta caagcgccag gcacagactg accgagtgag cctgcggaac 240 ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag gatgtacggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggcatg accagtccgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg agctcctgga ccgcggcgga cacggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagtggag agcctacctg 480 gagggcctgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcgcggacc ccccaaagac acatgtgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agatagaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagcca 828 <210> 126 <211> 828 <212> DNA <213> Homo sapiens <400> 126 60. ggctcccact ccatgaggta tttccacacc gccatgtccc ggcccggccg cggggagccc cgcttcatca ccgtgggcta cgtggacgac acgctgttcg tgaggttcga cagcgacgcc 120 acgagtccga ggaggagcc gcggggcgcca tggatagc aggaggggcc ggagtattgg gaccgggaga cacagatctc caagaccaac acacagactt accgagagag cctgcggaac ctgcgcggct gctacaacca gagcgaggcc gggtctcaca cttggcagag gatgtatggc tgcgacctgg ggcccgacgg gcgcctcctc cgcgggtata accagttagc ctacgacggc 360 aaggattaca tcgccctga cgaggacctg agctcctgga ccgcggcgga caccgcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagctgag agcctacctg 480 gagggcctgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 600. cgcgcggacc ccccaaagac acatgtgacc cccccc tctctgacc tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agatagaacc ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagcca 828 <210> 127 <211> 828 <212> DNA <213> Homo sapiens <400> 127 60. ggctcccact ccatgaggta tttctacacc gccatgtccc ggcccggccg cggggagccc cgcttcatcg cagtgggcta cgtggacgac acccagttcg tgaggttcga cagcgacgcc 120 gcgagtccga ggatggcgcc ccggggcgcca tggatagc aggaggggcc ggagtattgg 180 gacggggaga cacggaacat gaaggcctcc gcgcagactt accgagagaa cctgcggatc 240 gcgctccgct gctacaacca gagcgaggcc gggtctcaca tcatccaggt gatgtatggc 300 tgcgacgtgg ggccggacgg gcgcctcctc cgcgggcatg accagtccgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg agctcctgga ccgcggcgga cacggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagctgag agcctacctg 480 gagggcctgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcgcggacc ccccaaagac acatgtgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca gaccagcagg agatagaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc aaagcccctc accctgagat gggagcca 828 <210> 128 <211> 828 <212> DNA <213> Homo sapiens <400> 128 60. ggctcccact ccatgaggta tttctacacc gccatgtccc ggcccggccg cggggagccc cgcttcatcg cagtgggcta cgtggacgac acccagttcg tgaggttcga cagcgacgcc 120 gcgagtccga ggacggagcc ccggggcgcca tggatagc aggaggggcc ggagtattgg 180 gacggggaga cacggacat gaaggcctcc gcgcagactt accgagaga cctgcggatc gcgctccgct gctacaacca gagcgaggcc gggtctcaca tcatccagag gatgtatggc tgcgacctgg ggcccgacgg gcgcctcctc cgcgggcatg accagtccgc ctacgacggc 360 aaggattaca tcgccctga cgaggacctg agctcctgga ccgcggcgga caccgcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagctgag agcctacctg 480 gagggcctgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 600. cgcgcggacc ccccaaagac acacgtgacc cccaccccg tctctgacca tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga cactgagctt gtggagacca gaccagcagg agatagaacc ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacatgccat 780 gtacagcatg aggggctgcc gaagcccctc accctgagat gggagcca 828 <210> 129 <211> 828 <212> DNA <213> Homo sapiens <400> 129 tgctcccact ccatgaggta tttctacacc gctgtgtccc ggcccagccg cggagagccc cacttcatcg cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagtccaa gaggggagcc gcggggcgccg tgggtggagc aggaggggcc ggagtattgg 180 gaccgggaga cacagaagta caagcgccag gcacagactg accgagtgaa cctgcggaaa 240 ctacgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag gatgtacggc 300 tgcgacctgg ggcccgacgg gcgcctcctc cgcgggtatg accagtccgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcctgga ccgccgcgga cacagcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagtggag agcctacctg 480 gagggcgagt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcgcggaac acccaaagac acacgtgacc caccatcccg tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct acggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca ggccagcagg agatggaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacgtgccat 780 gtgcagcacg aggggctgcc ggagcccctc accctgagat gggagcca 828 <210> 130 <211> 828 <212> DNA <213> Homo sapiens <400> 130 ggctcccact ccatgaggta tttctacacc gctgtgtccc ggcccggccg cggggagccc cacttcatcg cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagtccga gaggggagcc gcggggcgccg tgggtggagc aggaggggcc ggagtattgg 180 gaccgggaga cacagaagta caagcgccag gcacagactg accgagtgag cctgcggaac ctgcgcggct gctacaacca gagcgaggcc gggtctcaca tcatccagag gatgtatggc tgcgacgtgg ggcccgacgg gcgcctcctc cgcgggtatg accagtacgc ctacgacggc 360 aaggattaca tcgccctga cgaggatctg cgctcctgga ccgccgcgga cacggcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagctgag agcctacctg 480 gagggcctgt gcgtggagtg gctccgcaga tacctgaaga atgggaagga gacgctgcag 540 600. cgcgcggac accccaaagac acacgtgacc caccatcccg tctctgacca tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagtgggat 660 ggggaggacc aaactcagga cactgagctt gtggagacca ggccagcagg agatggaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacgtgccat 780 gtgcagcacg aggggctgcc ggagcccctc accctgagat gggagccg 828 <210> 131 <211> 828 <212> DNA <213> Homo sapiens <400> 131 tgctcccact ccatgaggta tttctacacc gccgtgtccc ggcccggccg cggagagccc cgcttcatcg cagtgggcta cgtggacgac acgcagttcg tgcagttcga cagcgacgcc 120 gcgagtccaa gaggggagcc gcggggcgccg tgggtggagc aggaggggcc ggagtattgg 180 gaccgggaga cacagaagta caagcgccag gcacagactg accgagtgaa cctgcggaaa 240 ctgcgcggct gctacaacca gagcgaggcc gggtctcaca ccctccagag gatgtatggc 300 tgcgacctgg ggcccgacgg gcgcctcctc cgcgggtata accagttcgc ctacgacggc 360 aaggattaca tcgccctgaa tgaggacctg cgctcctgga ccgccgcgga caaggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgtgaggcgg agcagcggag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagaa gacgctgcag 540 cgcgcggaac acccaaagac acacgtgacc caccatcccg tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggcgaggacc aaactcagga caccgagctt gtggagacca ggccagcagg agatggaacc 720 ttccagaagt gggcagctgt ggtggtgcct tctggagaag agcagagata cacgtgccat 780 gtgcagcacg aggggctgcc agagcccctc accctgagat gggggcca 828 <210> 132 <211> 828 <212> DNA <213> Homo sapiens <400> 132 tgctcccact ccatgaggta tttcgacacc gccgtgtccc ggcccggccg cggagagccc cgcttcatct cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagtccga gaggggagcc gcggggcgccg tgggtggagc aggaggggcc ggagtattgg 180 gaccggggaga cacagaacta caagcgccag gcacaggctg accgagtgag cctgcggaac ctgcgcggct gctacaacca gagcgaggac gggtctcaca ccctccagag gatgtatggc tgcgacctgg ggcccgacgg gcgcctcctc cgcgggtatg accagtccgc ctacgacggc 360 aaggattaca tcgccctga cgaggacctg cgctcctgga ccgccgcgga caccgcggct cagatcaccc agcgcaagtt ggaggcggcc cgtgcggcgg agcagctgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 600. cgcgcagac ccccaaagac acacgtgacc ccccccc tctctgacca tgaggccacc ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga caccgagctt gtggagacca ggccagcagg agatggaacc ttccagaagt gggcagctgt ggtggtgcct tctggacaag agcagagata cacgtgccat 780 atgcagcacg aggggctgca agagcccctc accctgagct gggagcca 828 <210> 133 <211> 828 <212> DNA <213> Homo sapiens <400> 133 60. ggctcccact ccatgaggta tttctacacc tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccaggaga cacggaatgt gaaggcccag tcacagactg accgagtgga cctggggacc 240 ctgcgcggct gctacaacca gagcgaggac ggttctcaca ccatccagat aatgtatggc 300 tgcgacgtgg ggccggacgg gcgcttcctc cgcgggtacc ggcaggacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcttgga ccgcggcgga catggcagct 420 cagatcacca agcgcaagtg ggaggcggcc catgcggcgg agcagcagag agcctacctg 480 gagggccggt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcggctgt ggtggtgcct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagctg 828 <210> 134 <211> 828 <212> DNA <213> Homo sapiens <400> 134 60. ggctcccact ccatgaggta tttctccaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gacgaggaga gaggaagt gaggcccac tcacagactg accgagaga cctgcggatc 300. gcgctccgct gctacaacca gagcgaggcc ggttctcaca ccctccagat gatgtttggc tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtacc accagtacgc ctacgacggc 360 aaggattaca tcgccctgaa agaggacctg cgctcttgga ccgcggcgga catggcggct cagatcaccc agcgcaagtg ggaggcggcc cgtgtggcgg agcagttgag agcctacctg 480 gagggcacgt gcgtggacgg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccact 600 ctgagatgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctt gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcagctgt ggtggtacct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagcca 828 <210> 135 <211> 828 <212> DNA <213> Homo sapiens <400> 135 ggctcccact ccatgaggta tttctccaca tccgtgtccc ggcccggcag tggagagccc 60 cgcttcatcg cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcgggcgccg tggatagagc aggagaggcc tgagtattgg 180 gaccaggaga cacggaatgt gaaggcccag tcacagactg accgagtgga cctggggacc 240 ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat aatgtatggc 300 tgcgacgtgg ggtcggacgg gcgcttcctc cgcgggtatg aacagcacgc ctacgacggc 360 aaggattaca tcgccctgaa cgaggacctg cgctcttgga ccgcggcgga catggcggct 420 cagatcaccc agcgcaagtg ggaggcggcc cgttgggcgg agcagttgag agcctacctg 480 gagggcacgt gcgtggagtg gctccgcaga tacctggaga acgggaagga gacgctgcag 540 cgcacggacc cccccaagac acatatgacc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cactgacctg gcagcgggat 660 ggggaggacc agacccagga cacggagctc gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggcggctgt ggtggtgcct tctggagagg agcagagata cacctgccat 780 gtgcagcatg agggtctgcc caagcccctc accctgagat gggagctg 828 <210> 136 <211> 828 <212> DNA <213> Homo sapiens <400> 136 60. ggctcccact ccatgaggta tttcaccaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg ccgtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc 120 gcgagccaga ggatggagcc gcggggcgccg tggatagc aggaggggcc ggagtattgg 180 gaccggaca cacggatgt gaggcccac tcacagattg accgagtgga cctggggacc ctgcgcggct gctacaacca gagcgaggcc ggttctcaca ccatccagat gatgtatggc tgcgacgtgg ggtcggacgg gcgcttc...

Claims

1. A polynucleotide molecule comprising, in the 5' to 3' direction, (i) a first universal target sequence, (ii) a nucleotide sequence encoding an antigenic peptide, (iii) a second universal target sequence different from the first universal target sequence, (iv) a beta 2 microglobulin (β2M) sequence, and (v) a multiple histocompatibility complex (MHC) allele sequence.

2. 2. The polynucleotide of claim 1, comprising, in a 5' to 3' direction, (i) a promoter sequence, (ii) a first universal target sequence comprising the sequence set forth in SEQ ID NO:3, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target sequence comprising the sequence set forth in SEQ ID NO:4, (v) a β2M sequence comprising the sequence set forth in SEQ ID NO:106, (vi) an MHC allele sequence comprising a sequence selected from the group consisting of sequences set forth in SEQ ID NOs:109-174, (vii) a first affinity tag sequence comprising the sequence set forth in SEQ ID NO:29, (viii) a protease cleavage site sequence comprising the sequence set forth in SEQ ID NO:31, and (ix) a second affinity tag sequence comprising the sequence set forth in SEQ ID NO:

35.

3. The polynucleotide of claim 1 , wherein the antigenic peptide is selected from the group consisting of a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phosphoantigen, and a microbial antigen.

4. The polynucleotide of claim 1 , wherein the antigenic peptide is a neoantigen.

5. The polynucleotide of claim 4, wherein the neoantigen is selected by analyzing tumor sequencing data from a subject to identify one or more somatic mutations.

6. The polynucleotide of claim 5, wherein the analysis is performed using an in silico prediction algorithm.

7. The polynucleotide of claim 6, wherein the predictive algorithm further comprises an MHC binding algorithm for predicting binding between neoantigens and MHC alleles.

8. 8. The polynucleotide of claim 1, wherein the MHC allele is a mammalian MHC allele.

9. 9. The polynucleotide of claim 1, wherein the MHC allele is a human MHC allele.

10. The polynucleotide of claim 8 , wherein the MHC allele is a class I HLA allele.

11. The polynucleotide of claim 10, wherein the HLA allele comprises an HLA-A, HLA-B, or HLA-C allele.

12. The polynucleotide of claim 10, wherein the HLA allele is an HLA allele of a subject.

13. HLA alleles are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:0 1, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A* 68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HL A-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01 , HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*3 8:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA- 11. The polynucleotide of claim 10, wherein the polynucleotide is selected from the group consisting of HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:

01.

14. The polynucleotide of claim 10, wherein the HLA allele comprises a sequence selected from the group consisting of SEQ ID NOs: 109-174.

15. 15. A polynucleotide according to any one of claims 1 to 14, wherein the β2M allele is a mammalian β2M allele.

16. The polynucleotide of claim 15, wherein the β2M allele is a human β2M allele.

17. The polynucleotide of claim 16, wherein the β2M allele comprises the sequence set forth in SEQ ID NO:

106.

18. 18. The polynucleotide of any one of claims 1 to 17, wherein the nucleotide sequence encoding the antigenic peptide is 20-70, 20-30, 25-35, 20-45, 30-45, 40-70, or 45-70 nucleotides in length.

19. The polynucleotide according to any one of claims 1 to 18, wherein the nucleotide sequence encoding the antigenic peptide is 20 to 30 nucleotides in length.

20. 20. The polynucleotide of any one of claims 1 to 19, wherein the first universal target sequence is 4-50, 4-15, 15-40, 15-35, 15-30, 20-40, 25-40, or 30-40 nucleotides in length.

21. 21. The polynucleotide of any one of claims 1 to 20, wherein the first universal target sequence is 25 to 35 nucleotides in length.

22. 21. The polynucleotide of any one of claims 1 to 20, wherein the first universal target sequence is at least about 15 nucleotides in length.

23. 23. The polynucleotide of any one of claims 1 to 22, wherein the first universal target sequence comprises the sequence shown in SEQ ID NO:

3.

24. 24. The polynucleotide of any one of claims 1 to 23, wherein the second universal target sequence is 4 to 50, 4 to 15, 15 to 40, 15 to 35, 15 to 30, 20 to 40, 25 to 40, or 30 to 40 nucleotides in length.

25. 25. The polynucleotide of any one of claims 1 to 24, wherein the second universal target sequence is 25 to 35 nucleotides in length.

26. 25. The polynucleotide of any one of claims 1 to 24, wherein the second universal target sequence is at least about 15 nucleotides in length.

27. 27. The polynucleotide of any one of claims 1 to 26, wherein the second universal target sequence comprises the sequence set forth in SEQ ID NO:

4.

28. 28. The polynucleotide of any one of claims 1 to 27, wherein the first and second universal target sequences comprise polymerase chain reaction (PCR) primer target sequences.

29. 29. The polynucleotide of any one of claims 1 to 28, wherein the first and second universal target sequences comprise a restriction enzyme cleavage site.

30. 30. The polynucleotide of any one of claims 1 to 29, wherein the first universal target sequence further comprises a signal sequence.

31. 31. The polynucleotide of claim 30, wherein the signal sequence encodes a signal sequence comprising a human growth hormone signal sequence, a hIG1 kappa light chain signal sequence, a beta 2 microglobulin signal sequence, or an IL2 signal sequence.

32. 32. The polynucleotide of claim 31 , wherein the signal sequence comprises a sequence comprising the sequence set forth in SEQ ID NO: 1, 24, 26, or 28.

33. 31. The polynucleotide of claim 30, wherein the signal sequence encodes a human growth hormone (HGH) signal sequence.

34. 34. The polynucleotide of claim 33, wherein the signal sequence comprises the sequence shown in SEQ ID NO:

1.

35. 31. The polynucleotide of claim 30, wherein the signal sequence is 40-90, 40-60, 45-70, 50-80, 60-90, 55-70, 60-80, or 70-90 nucleotides in length.

36. 36. The polynucleotide of any one of claims 1 to 35, wherein the 3' end of the polynucleotide sequence further comprises a purification cluster sequence comprising, in the 5' to 3' direction, (i) a first affinity tag sequence, (ii) a protease cleavage site sequence, and (iii) a second affinity tag sequence.

37. 37. The polynucleotide of claim 36, wherein the first and second affinity tags are selected from the group consisting of AviTag, streptavidin-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and Myc-tag.

38. 37. The polynucleotide of claim 36, wherein the first affinity tag comprises a sequence comprising the sequence set forth in SEQ ID NO:29 and the second affinity tag comprises a sequence comprising the sequence set forth in SEQ ID NO:

33.

39. 37. The polynucleotide of claim 36, wherein the protease cleavage site sequence is selected from the group consisting of a TEV cleavage site sequence, a thrombin cleavage site sequence, a factor Xa cleavage site sequence, an enteropeptidase cleavage site sequence, and a rhinovirus 3C protease cleavage site sequence.

40. 40. The polynucleotide of claim 39, wherein the protease cleavage site sequence comprises the TEV cleavage site sequence shown in SEQ ID NO:

31.

41. 40. The polynucleotide of claim 39, wherein the first affinity tag sequence comprises the sequence set forth in SEQ ID NO:29, the protease cleavage site comprises the sequence set forth in SEQ ID NO:31, and the second affinity tag comprises the sequence set forth in SEQ ID NO:

33.

42. 42. The polynucleotide of any one of claims 1 to 41, wherein the second universal target sequence further comprises a first linker sequence.

43. 43. The polynucleotide of claim 42, wherein the first linker sequence comprises the sequence set forth in SEQ ID NO: 10, 14, 16, or 18.

44. 44. The polynucleotide of any one of claims 1 to 43, further comprising a second linker sequence between the β2M sequence and the MHC allele sequence.

45. 45. The polynucleotide of claim 44, wherein the second linker sequence comprises the sequence shown in SEQ ID NO: 10 or 20.

46. 46. ​​The polynucleotide of any one of claims 1 to 45, further comprising a third linker sequence between the MHC allele sequence and the first affinity tag.

47. 47. The polynucleotide of claim 46, wherein the third linker sequence comprises the sequence shown in SEQ ID NO: 12 or 22.

48. 48. The polynucleotide of any one of claims 1 to 47, wherein the 5' end of the polynucleotide sequence further comprises a promoter sequence linked to the 5' end of the first universal target.

49. 49. The polynucleotide of claim 48, wherein the promoter sequence is selected from the group consisting of CMV, EF1α, and SV40 promoters.

50. 49. The polynucleotide of claim 48, wherein the promoter sequence is a CMV promoter sequence.

51. 51. The polynucleotide of claim 50, wherein the promoter sequence is a CMV promoter sequence.

52. 52. The polynucleotide of any one of claims 1 to 51, wherein the 3' end of the polynucleotide sequence further comprises a polyA sequence.

53. 53. The polynucleotide of claim 52, wherein the polyA sequence is selected from a bGH polyA sequence, an SV40 polyA sequence, an hGH polyA sequence, and an rbGlob polyA sequence.

54. 53. The polynucleotide of claim 52, wherein the polyA sequence comprises the bGH polyA sequence set forth in SEQ ID NO:

179.

55. An expression construct comprising a polynucleotide molecule according to any one of claims 1 to 54.

56. The construct of claim 55, wherein the expression construct comprises a plasmid or a viral vector.

57. 57. A host cell comprising a polynucleotide molecule or expression construct according to any one of claims 1 to 56.

58. 58. The cell of claim 57, wherein the polynucleotide is integrated into the cell genome.

59. 58. The host cell of claim 57, wherein the polynucleotide is extrachromosomal.

60. 60. The cell of any one of claims 57 to 59, wherein the host cell is a mammalian cell.

61. 61. The cell of claim 60, which is a human cell.

62. 61. The cell of any one of claims 57 to 60, selected from the group consisting of a stem cell, a tumor cell, an immortalized cell, and a fetal cell.

63. 60. The cell of any one of claims 57 to 59, wherein the host cell is a prokaryotic cell.

64. 64. The cell of claim 63, which is an Escherichia coli cell.

65. 65. A cell according to any one of claims 57 to 64, which expresses a BirA protein or a fragment thereof.

66. 66. A library comprising the composition of any one of claims 1 to 65, wherein the library comprises two or more different polynucleotide molecules, each different polynucleotide molecule comprising (i) a first universal sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is non-identical for each of the two or more polynucleotide molecules, (iii) a second universal target sequence, (iv) a β2M sequence, and (v) an MHC allele sequence.

67. 67. The library of claim 66, wherein the MHC allele sequences are not identical for each of the two or more polynucleotide molecules.

68. 68. The library of claim 66 or 67, comprising between 20 and 500 different polynucleotide molecules.

69. 68. The library of claim 66 or 67, comprising at least 66 different polynucleotide molecules.

70. At least HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HL A-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68: 02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA- B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01 , HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B* 38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, H 70. The library of claim 69, comprising HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01 alleles.

71. The library according to claim 69, comprising at least the sequences set forth in SEQ ID NOs: 109 to 174.

72. A polypeptide comprising, from the amino terminus to the carboxyl terminus, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide.

73. 73. The polypeptide of claim 72, comprising, in the 5' to 3' direction, (i) a promoter peptide comprising the sequence set forth in SEQ ID NO:2, (ii) a first universal target peptide, (iii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a tumor neo-antigen, (iv) a second universal target peptide comprising the sequence set forth in SEQ ID NO:15 or 17, (v) a β2M peptide comprising the sequence set forth in SEQ ID NO:105, (vi) an MHC peptide comprising the sequence set forth in SEQ ID NO:38-103, (vii) a first affinity peptide comprising the sequence set forth in SEQ ID NO:30, (viii) a protease cleavage site peptide comprising the sequence set forth in SEQ ID NO:32, and (ix) a second affinity peptide comprising the sequence set forth in SEQ ID NO:

36.

74. 73. The polypeptide of claim 72, wherein the antigenic peptide is selected from the group consisting of a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phosphoantigen, and a microbial antigen.

75. 75. The polypeptide of claim 72 or 74, wherein the antigenic peptide is a neoantigen.

76. 76. The polypeptide of any one of claims 72 to 75, wherein the neoantigen is selected by analyzing tumor sequencing data from a subject to identify one or more somatic mutations.

77. 77. A polypeptide according to any one of claims 72 to 76, wherein the analysis is carried out using an in silico prediction algorithm.

78. 78. The polypeptide of any one of claims 72 to 77, wherein the predictive algorithm further comprises an MHC binding algorithm for predicting binding between a neo-antigen and an MHC peptide.

79. 79. The polypeptide of any one of claims 72 to 78, wherein the MHC peptide is a mammalian MHC peptide.

80. 79. The polypeptide of any one of claims 72 to 78, wherein the MHC peptide is a human MHC peptide.

81. 80. The polypeptide of any one of claims 72 to 79, wherein the MHC peptide is a class I HLA peptide.

82. 82. The polypeptide of any one of claims 72 to 81, wherein the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.

83. 83. The polypeptide of claim 82, wherein the HLA peptide is an HLA peptide of a subject.

84. The HLA peptides are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33: 01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA- A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01 , HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B* 42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, H LA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05 83. The polypeptide of claim 82, comprising HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA-C*17:

01.

85. 83. The polypeptide of claim 82, wherein the HLA peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 38-103.

86. 86. A polypeptide according to any one of claims 72 to 85, wherein the HLA peptide comprises a Y84A or Y84C mutation.

87. 86. A polypeptide according to any one of claims 72 to 85, wherein the β2M peptide is a mammalian β2M peptide.

88. 88. The polypeptide of claim 87, wherein the β2M peptide is a human β2M peptide.

89. The polypeptide of claim 88, wherein the β2M peptide comprises the sequence set forth in SEQ ID NO:

105.

90. 89. The polypeptide of claim 88, wherein the β2M peptide comprises the sequence set forth in SEQ ID NO: 107, which comprises an S88C mutation at amino acid 88.

91. 91. The polypeptide of any one of claims 72 to 90, wherein the antigenic peptide is 7 to 15 amino acids, 7 to 10, 8 to 9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

92. 92. The polypeptide of any one of claims 72 to 91, wherein the first universal target peptide further comprises a signal peptide.

93. 93. The polypeptide of claim 92, wherein the signal peptide comprises a human growth hormone signal peptide, a hIG1 kappa light chain signal peptide, a beta 2 microglobulin signal peptide, or an IL2 signal peptide.

94. 94. The polypeptide of claim 93, wherein the signal peptide comprises a sequence comprising the sequence set forth in SEQ ID NO: 2, 23, 25, or 27.

95. 94. The polypeptide of claim 93, wherein the signal peptide comprises the human growth hormone (HGH) signal peptide sequence shown in SEQ ID NO:

2.

96. 97. The polypeptide of any one of claims 72 to 96, wherein the signal sequence is 15-45, 15-30, 20-45, 20-30, or 30-45 amino acids in length.

97. 97. The polypeptide of any one of claims 72 to 96, wherein the carboxyl terminus of the polypeptide further comprises a purification cluster comprising, from the amino terminus to the carboxyl terminus, (i) a first affinity peptide, (ii) a protease cleavage site, and (iii) a second affinity peptide.

98. The polypeptide of claim 97, wherein the first and second affinity peptides are selected from the group consisting of AviTag, streptavidin-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and Myc-tag.

99. The polypeptide of claim 98, wherein the first affinity peptide comprises an array comprising SEQ ID NO: 30 and the second affinity peptide comprises an array comprising SEQ ID NO:

34.

100. 98. The polypeptide of claim 97, wherein the protease cleavage site is selected from the group consisting of a TEV cleavage site, a thrombin cleavage site, factor Xa, an enteropeptidase cleavage site, and a rhinovirus 3C protease cleavage site.

101. The polypeptide of claim 100, wherein the protease cleavage site comprises the TEV cleavage site sequence shown in SEQ ID NO:

32.

102. 98. The polypeptide of claim 97, wherein the purification cluster comprises an AviTag peptide sequence as set forth in SEQ ID NO: 30, a TEV cleavage site sequence as set forth in SEQ ID NO: 32, and a His6 peptide sequence as set forth in SEQ ID NO: 34 or 36.

103. 103. The polypeptide of claim 102, wherein the purification cluster comprises two or more copies of the His6 peptide sequence shown in SEQ ID NO:

34.

104. 98. The polypeptide of claim 97, wherein the purification cluster comprises the sequence shown in SEQ ID NO:

37.

105. 105. The polypeptide of any one of claims 72 to 104, wherein the second universal target peptide further comprises a linker comprising the sequence set forth in SEQ ID NO: 9, 11, 15, or 17.

106. 106. The polypeptide of any one of claims 72 to 105, further comprising a second linker sequence between the β2M sequence and the MHC allele sequence.

107. The polypeptide of claim 106, wherein the second linker sequence comprises the sequence shown in SEQ ID NO: 9 or 19.

108. 108. The polypeptide of any one of claims 72 to 107, further comprising a third linker sequence between the MHC allele sequence and the first affinity tag.

109. The polypeptide of claim 108, wherein the third linker sequence comprises the sequence shown in SEQ ID NO: 13 or 21.

110. 110. A polypeptide according to any one of claims 72 to 109, which is biotinylated.

111. 111. A library comprising the polypeptides of claims 72-110, wherein the library comprises two or more different polypeptide molecules, the different polypeptide molecules comprising (i) a first universal target peptide, (ii) an antigenic peptide, where the antigenic peptide is not identical to each of the two or more polypeptide molecules, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide.

112. The library of claim 111, wherein the MHC peptide is not identical for each of the two or more polypeptide molecules.

113. 113. The library of claim 111 or 112, comprising from 20 to 500 different polypeptides.

114. 113. The library of claim 111 or 112, comprising at least 66 different polypeptides.

115. HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A* 68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, H LA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*5 0:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HL A-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38 :01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA 115. The library of claim 114, comprising HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01 polypeptides.

116. 116. The polypeptide of claims 72-115, wherein the polypeptide is bound to a particle, the particle being selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer.

117. The polypeptide of claim 116, which is attached to the particle via a linker.

118. 118. The polypeptide of claim 116 or 117, wherein the nanoparticle is a magnetic nanoparticle or a polystyrene nanoparticle.

119. The polypeptide of claim 118, wherein the magnetic nanoparticle comprises magnetic iron oxide.

120. 117. The polypeptide of claim 116, wherein the bead is an agarose bead or a sepharose bead.

121. 121. The polypeptide of any one of claims 116 to 120, further comprising a fluorophore.

122. 122. The polypeptide of claim 121, wherein the fluorophore is attached to a particle, with or without a linker.

123. A library comprising a polypeptide described in any one of claims 116 to 122, wherein the library comprises two or more different polypeptide molecules, the antigenic peptide is not identical for each of the two or more polypeptide molecules, and each different polypeptide is bound to a particle.

124. The library of claim 123, wherein the MHC peptide is not identical for each of the two or more polypeptide molecules.

125. 125. The library of claim 123 or 124, further comprising a unique defined barcode sequence operably associated with the identity of each distinct polypeptide.

126. 126. The library of claim 125, comprising between 20 and 500 different polypeptides.

127. 126. The library of claim 125, comprising at least 66 different polypeptides.

128. 128. A kit comprising the composition of any one of claims 1 to 127 and instructions for use.

129. 1. A method for producing a polynucleotide molecule, comprising: a. obtaining a first polynucleotide sequence comprising, in a 5' to 3' direction, (i) a first universal target sequence comprising a restriction enzyme recognition site, (ii) a second universal target sequence comprising a restriction enzyme recognition site that is not identical to the first universal target sequence, (iii) a β2M sequence, and (iv) an MHC allele sequence; b. obtaining a second polynucleotide that includes, in a 5' to 3' direction, (i) a portion of the first universal target sequence, (ii) a sequence encoding an antigenic peptide, and (iii) a portion of the second universal target sequence; c. obtaining a third polynucleotide that includes, in the 5' to 3' direction, (i) a reverse complement of a portion of the second universal target sequence, (ii) a reverse complement of a sequence encoding an antigenic peptide, and (iii) a reverse complement of a portion of the first universal target sequence; d. mixing the second and third polynucleotides together such that complementary sequences anneal; e. performing a restriction digest on the first polynucleotide sequence with at least one restriction enzyme; and f. Ligating the first and second polynucleotides together by mixing the digested first polynucleotide and the annealed second and third polynucleotides with DNA ligation reaction reagents. The method includes:

130. 130. The method of claim 129, further comprising phosphorylating the 5' nucleotide of the second and third polynucleotides after the complementary sequences have annealed, wherein phosphorylating comprises incubating the first or the second and third polynucleotides with an enzyme.

131. 131. The method of claim 130, wherein the enzyme is T4 kinase.

132. 132. The method of any one of claims 129 to 131, wherein the first polynucleotide further comprises a promoter sequence.

133. 133. The method of claim 132, wherein the promoter sequence is selected from the group consisting of CMV, EF1α, and SV40 promoters.

134. 134. The method of claim 133, wherein the promoter sequence is CMV.

135. 135. The method of any one of claims 129 to 134, wherein the 3' end of the second polynucleotide sequence further comprises a purification cluster sequence comprising, in a 5' to 3' direction, (i) a first affinity tag sequence, (ii) a protease cleavage site sequence, and (iii) a second affinity tag sequence.

136. 136. The method of any one of claims 129 to 135, wherein the 3' end of the second polynucleotide sequence further comprises a polyA sequence.

137. 137. The method of any one of claims 129 to 136, wherein the first polynucleotide is an expression construct.

138. 137. The method of any one of claims 129 to 136, further comprising inserting the produced polynucleotide into an expression construct.

139. 139. The method of any one of claims 129 to 138, wherein obtaining the third and fourth polynucleotide sequences further comprises obtaining a predictive dataset of tumor sequencing data from the subject.

140. 140. The method of claim 139, wherein obtaining the third and fourth polynucleotide sequences further comprises predicting sequences encoding antigenic peptides from tumor sequencing data.

141. 141. The method of claim 139 or 140, wherein the steps of obtaining the third and fourth polynucleotide sequences further comprise synthesizing a polynucleotide encoding an antigenic peptide based on the predicted sequences.

142. 142. The method of any one of claims 129-141, wherein the method further comprises producing a library comprising two or more different polynucleotide molecules, the different polynucleotide molecules comprising: (i) a first universal target sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the antigenic peptide is a non-identical nucleotide sequence for each of the two or more polypeptide molecules, (iii) a second universal target sequence that is different from the first universal target sequence, (iv) a β2M peptide, and (v) an MHC allele sequence.

143. 143. The method of claim 142, wherein the MHC alleles are not identical for each of the two or more polynucleotide molecules.

144. 144. The method of claim 143, wherein the library comprises between 20 and 500 different polynucleotide molecules.

145. 144. The method of claim 143, wherein the library comprises at least 66 different polynucleotide molecules.

146. 145. The method of any one of claims 129 to 144, wherein the first polynucleotide is an expression construct.

147. 145. The method of any one of claims 129 to 144, further comprising inserting the produced polynucleotide into an expression construct.

148. 147. The method of claim 146, wherein the method further comprises producing a library comprising two or more expression constructs comprising different polynucleotide molecules, the different polynucleotide molecules comprising: (i) a first universal target sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is not identical for each of the two or more polynucleotide molecules, (iii) a second universal target sequence that is different from the first universal target sequence, (iv) a β2M sequence, and (v) an MHC allele sequence.

149. 149. The method of claim 148, wherein the MHC alleles are not identical for each of the two or more polynucleotide molecules.

150. 150. The method of claim 149, wherein the MHC allele is selected from the sequence set forth in the group consisting of SEQ ID NOs: 109-174.

151. 151. The method of any one of claims 129 to 150, further comprising expressing a polyprotein molecule from the polynucleotide molecule.

152. 152. The method of claim 151, wherein the polynucleotide is transfected or transduced into the cell.

153. 153. The method of claim 152, wherein the polynucleotide is integrated into the cell genome.

154. 153. The method of claim 152, wherein the polynucleotide remains extrachromosomal in the cell.

155. 155. A method according to any one of claims 152 to 154, wherein the cell is a mammalian cell, optionally wherein the cell is a tumor cell line.

156. 155. The method of any one of claims 152 to 154, wherein the cell is a prokaryotic cell.

157. 157. The method of claim 156, wherein the cell is an Escherichia coli cell.

158. 156. The method of any one of claims 151 to 155, further comprising quantifying expression of the polyprotein.

159. The method of claim 158, wherein a non-expressed polyprotein sequence is used to improve the antigen peptide prediction analysis process of claim 140.

160. 159. The method of any one of claims 146 to 158, further comprising biotinylating the polyprotein.

161. The method of claim 160, wherein the polyprotein is biotinylated with the BirA protein.

162. 161. The method of any one of claims 146 to 160, further comprising purifying the polyprotein.

163. 163. The method of claim 162, wherein the purification step comprises affinity chromatography.

164. 164. The method of claim 163, wherein the affinity chromatography comprises immobilized metal affinity chromatography comprising a support, a chelating agent, and a divalent metal.

165. 165. The method of claim 164, wherein the chelating agent for affinity chromatography is nitrolotriacetic acid (NTA) or iminidiacetic acid (IDA).

166. 165. The method of claim 164, wherein the divalent metal is selected from the group consisting of nickel (Ni), cobalt (Co), copper (Cu), and iron (Fe).

167. 165. The method of claim 164, wherein the chelating agent is NTA and the divalent metal is Ni.

168. 168. The method of any one of claims 164 to 167, wherein the support is selected from the group consisting of agarose beads, sepharose beads, and magnetic beads.

169. 169. The method of any one of claims 160 to 168, further comprising quantifying the biotinylation level of the purified polyprotein.

170. 170. The method of any one of claims 162 to 169, further comprising binding the polypeptide to at least one particle, the particle being selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer.

171. 171. The method of claim 170, wherein the nanoparticles are magnetic nanoparticles or polystyrene nanoparticles.

172. 172. The method of claim 171, wherein the magnetic nanoparticles comprise magnetic iron oxide.

173. 171. The method of claim 170, wherein the beads are agarose beads or sepharose beads.

174. 174. The method of any one of claims 170 to 173, wherein the particle further comprises a fluorophore.

175. 175. The method of claim 174, wherein the fluorophore is attached to at least one particle, with or without a linker.

176. 176. The method of any one of claims 151-175, further comprising generating a library comprising two or more different polyproteins bound to at least one particle, wherein the different polypeptides comprise: (i) a first universal target peptide, (ii) an antigenic peptide, where the antigenic peptide is not identical for each of the two or more polypeptide molecules, (iii) a second universal target peptide that is different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide.

177. 177. The method of claim 176, wherein the MHC peptide is not identical for each of the two or more polypeptide molecules.

178. 178. The method of claim 177, wherein the MHC peptide is selected from the sequence set forth in the group consisting of SEQ ID NOs: 39-104.

179. 178. The method of claim 176 or 177, wherein the library comprises between 20 and 500 different single polyproteins.

180. 178. The method of claim 176 or 177, wherein the library comprises at least 66 different single polyproteins.

181. 1. A method for producing a polynucleotide molecule, comprising: a. obtaining a first polynucleotide sequence comprising, in a 5' to 3' direction, (i) a first universal target sequence comprising a restriction enzyme recognition site, (ii) a second universal target sequence comprising a restriction enzyme recognition site that is not identical to the first target sequence, (iii) a β2M sequence, and (iv) an MHC allele sequence; b. obtaining a second polynucleotide sequence comprising, in a 5' to 3' direction, (i) a first universal target sequence, (ii) a sequence encoding an antigenic peptide, and (iii) a second universal target sequence; c. performing a restriction digest on the first polynucleotide sequence with at least one restriction enzyme; d. performing a restriction digest on the second polynucleotide sequence with at least one restriction enzyme; and e. ligating the first and second polynucleotides together by mixing the digested first and second polynucleotides together with DNA ligation reaction reagents. The method includes:

182. 1. A method for producing a polynucleotide molecule, comprising: a. obtaining a first polynucleotide sequence comprising a first universal target sequence; b. obtaining a second polynucleotide sequence comprising, in the 5' to 3' direction, (i) a second universal target sequence, (ii) a β2M sequence, and (iii) an MHC allele sequence; c. obtaining a third polynucleotide sequence comprising, in a 5' to 3' direction, a sequence encoding an antigenic peptide and a second universal target sequence; d. obtaining a fourth polynucleotide sequence comprising, in the 5' to 3' direction, a reverse complement of the antigenic peptide and a reverse complement of the first universal target sequence; e. combining the first, second, third, and fourth polynucleotides in solution; f. adding polymerase chain reaction (PCR) reagents; and g. performing a PCR reaction, wherein the complementary regions of the second universal target sequence in the second and third polynucleotides anneal to each other and the complementary regions of the first universal target sequence in the first and fourth polynucleotides anneal to each other; the annealed sequences provide priming sequences for the PCR extension and amplification reaction. The method includes:

183. 1. A method for isolating antigen-specific T cells, comprising: a. providing a polypeptide comprising, in an amino-terminal to carboxyl-terminal direction, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide, wherein the polypeptide is linked to a particle; b. Providing a sample known or suspected to contain one or more T cells; c. contacting the polypeptide with the sample, comprising providing conditions sufficient for a single T cell to bind to the particle-bound polypeptide; and d. Isolating a single T cell bound to the particle The method includes:

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