Antigen-binding proteins and use thereof

Abstract

Aspects of the present disclosure are directed to TCR-targeting polypeptides, including antibodies, antibody-drug conjugates, antibody fragments, antibody-like molecules, and chimeric receptors. Also disclosed herein are nucleic acids encoding for such TCR-targeting polypeptides and cells comprising such nucleic acids. Described are methods for treatment of cancer using TCR-targeting polypeptides.

Description

ANTIGEN-BINDING PROTEINS AND USE THEREOF

[0001] This application claims priority of U.S. Provisional Application No. 63 / 676,220 filed July 26, 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 24, 2025, is named ARCDP0851WO_sequence_listing.xml and is 99,759 bytes in size.BACKGROUNDI. Field of the Disclosure

[0003] Aspects of the disclosure relate to at least the fields of molecular biology and medicine.II. Background

[0004] The Vy9V52 TCR defines a major subset of human gamma delta (y5) T cells that play a unique role in bridging innate and adaptive immunity. Unlike conventional aP T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, y952 T cells can directly sense and respond to small molecular patterns expressed by stressed or infected cells in an MHC -unrestricted manner. The Vy9V52 TCR can be activated by the accumulation of small non-peptidic phosphoantigens (pAgs) upon dysregulation of the mevalonate or non-mevalonate isoprenoid biosynthesis pathways. Upon activation, y952 T cells rapidly produce cytokines like IFN-y and exhibit potent cytotoxicity against target cells through release of perforin, granzymes and engagement of death receptors (Vyborova et al., 2020). Overall, the Vy9V52 TCR enables a unique T cell population to serve as an early sensor of cellular insult and mediate rapid effector responses against a wide range of infections and malignancies, making it an attractive target for immunotherapeutic strategies. But there is a need in the art for compounds to activate these T cells.SUMMARY

[0005] To address certain needs in the art, the inventors have generated a diverse number of antigen-binding proteins, including Fabs and antibodies, that bind to antigens on gamma delta T cells, including y952 T cells. In certain aspects, the antigen-binding protein binds to a T cell receptor (TCR) on a y952 T cell, including a Vy9V52 TCR. The disclosure describes novel antibody and antigen binding fragments, as well as methods of using these antibodies and fragments. Also described are polypeptides comprising the antigen binding fragment(s) of the disclosure, and compositions comprising the polypeptides, antibodies, and / or antigen binding fragments of the disclosure. In certain aspsects, the antibody or antigen-binding fragment comprises a polypeptide capable of binding a public domain on a Vy9V52 TCR. In some aspects, the antibody or antigen-binding fragment comprises a polypeptide capable of binding the main chain and / or amino acid side chains of the public domain on a Vy9V52 TCR. The public domain of the Vy9V52 TCR can comprise a TLG tripeptide that is conserved across individuals. The antibody or antigen-binding domain may bind to the main chain of the Vy9V52 TCR at the TLG tripeptide and / or bind the TLG amino acid side chains. In certain aspects, the antibody or antigen-binding domain comprises a VXY tripeptide, where X is any amino acid, which can allow for binding of the public domain. In certain aspects, the antibody or antigenbinding fragment comprises a 3, 4, 5, 6, 7, 8, 9, 10 (or any range derivable therein) loop that can bind to the public domain, including the main chain of the public domain and / or the amino acid side chains.

[0006] Also described are nucleic acids encoding an antibody or antigen binding fragment of the disclosure. The disclosure also relates to an antibody comprising at least one polypeptide with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to one of the sequences of Table 1 or Table 2. Also described are nucleic acids, vectors, or expression vectors encoding antibodies of the disclosure and host cells comprising polypeptides, nucleic acids, vectors, antibodies, or antigen binding fragments of the disclosure. The nucleic acids of the disclosure may be DNA or RNA.

[0007] The disclosure also includes bi-specific antibodies, antigen-binding fragments, T cell engagers, and polypeptides that are capable of binding both a Vy9V52 TCR and a second antigen. In some aspects, the bi-specific antibodies, antigen-binding fragments, T cell engagers, and polypeptides comprise a first variable region that can bind a Vy9V52 TCR (including any of the antibodies of Table 1, or fragments or domains thereof) and a second variable region thatcan bind the second antigen. In some aspects, the second variable region comprises a variable region of a known antibody, including for example atezolizumab, belantamab, bevacizumab, blinatumomab, brentuximab, cetuximab, daratumumab, dinutuximab, elotuzumab, enfortumab, gemtuzumab, ibritumomab, inotuzumab, necitumumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pertuzumab, ramucirumab, rituximab, sacituzumab, or trastuzumab. In some aspects, the second variable region is capable of binding a differentiation antigen such as tyrosinase, TRP-1, TRP-2; a tumor-specific multilineage antigen such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; an overexpressed embryonic antigen such as CEA; an overexpressed oncogene and / or mutated tumor-suppressor gene such as p53, Ras, HER-2 / neu; a unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and / or a viral antigen, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alphafetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1 , CO-029, FGF-5, G250, Ga733\EpCAM, HTgp- 175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCASI, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, LI CAM, IL6, and MET.

[0008] In some aspects, the second variable region can bind a tumor antigen which includes, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD 19, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1, fucosyl GM1 , GloboH, MN- CA IX, EPC AM, EVT6- AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RUI, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M- CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1 , LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1 , VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, S SEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1 , MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG(TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171 , CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CX0RF61, folate receptor (FRa), folate receptor beta, R0R1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, mesothelin, and any combination thereof.

[0009] Further examples of tumor cell antigens to include at least 5T4, 8H9, avP6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD 19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CD133, CEA, c-Met, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPC AM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRa, GD2, G250 / CAIX, GD3, Glypi can-3 (GPC3), GUCY2C, HER1, HER2, ICAM-1, IL-13Ra2, IL-1 IRa, Kras, Kras G12D, L1CAM, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, MET, Mesothelin, Mucl, Mucl6, MUC18, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, SP17, Survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA- 125, ETA, Tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, EphA3, Telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-l / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmell7, TRP-1 / -2, P. polypeptide, MC1R, Prostate-specific antigen, P-catenin, BRCA1 / 2, CML66, Fibronectin, MART-2, TGF-PRII, WT-1, or VEGF receptors (e.g., VEGFR2), for example.

[0010] Also described is a method of a making a cell comprising transferring one or more nucleic acid(s) encoding an antibody of the disclosure into a cell. The method may comprise or further comprise culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid. The method may further comprise isolating the expressed polypeptide. The cell may be further defined as a human cell, B cell, T cell, Chinese hamster ovary, NS0 murine myeloma cell, PER.C6 cell, or a cell described herein.

[0011] The disclosure describes a method for treating or preventing a disease indicated for an immunotherapy in a patient, the method comprising administering to the patient an antibody, antigen binding fragment, polypeptide, nucleic acid, or host cell of the disclosure. Also described is a method for evaluating a sample from a patient, the method comprising contacting a biological sample from the patient, or extract thereof, with at least one antibody, antigen binding fragment, polypeptide, composition, or host cell of the disclosure. The antibodies,antigen binding fragments, or compositions of the disclosure may be used to treat a patient having a cancer, an infection, or any other disease that is indicated for an immunotherapy.

[0012] Methods include a method for treating or preventing cancer in a patient, the method comprising administering to the patient an antibody, antigen binding fragment, polypeptide, nucleic acid, or host cell of the disclosure. Also described is a method for evaluating a sample from a patient, the method comprising contacting a biological sample from the patient, or extract thereof, with at least one antibody, antigen binding fragment, or polypeptide, composition, or host cell of the disclosure. Also disclosed is a method for diagnosing cancer in a patient, the method comprising contacting a biological sample from the patient, or extract thereof, with at least one antibody, antigen binding fragment, composition, or polypeptide of any one of the disclosure. The antibodies, antigen binding fragments, or compositions of the disclosure may be used to treat a patient having cancer.

[0013] Provided by the disclosure is an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1. The disclosure also describes an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having or having at least 80% sequence identity or having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity with a HCDR1, HCDR2, and HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having or having at least least 80% sequence identity or having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity with a LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 may be determined from the variable region sequences by methods known in the art. The CDR may be a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 determined by the Chothia method. The CDR may be a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 determined by the Kabat method.The CDR may be a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 determined by the IMGT method.

[0014] The disclosure provides for an antibody or antigen binding fragment in which the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence that has at least 80% sequence identity to an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone. The HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 may each comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone. The HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 may each comprise the amino acid sequence of an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone.

[0015] The disclosure describes an antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1. The antibody or antigen binding fragment may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having or having at least 80% sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1. In some aspsects, the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3having the amino acid sequence of an of a HCDR1, HCDR2, and HCDR3 of a clone of Table 1 and the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 comprising the amino acid sequence of the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1.

[0016] The polypeptides of the disclosure may comprise at least two antigen binding fragments, wherein each antigen binding fragment is independently selected from an antigen binding fragment of the disclosure. The polypeptide may be multivalent. The polypeptide may be multispecific. The polypeptide may be bispecific. The polypeptide may comprise, comprise at least, or comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen binding regions. Each antigen binding region may be independently selected from an antigen binding region of the disclosure. The polypeptide may have repeated units of the same antigen binding region, such as at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeated units.

[0017] The heavy chain variable region may comprise an amino acid sequence with at least 80% sequence identity to a heavy chain variable region of an antibody clone of Table 1 and / or the light chain variable region may comprise an amino acid sequence with at least 80% sequence identity to the light chain variable region of the same antibody clone of Table 1. The heavy chain variable region may comprise an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to a heavy chain variable region of an antibody clone of Table 1 and / or the light chain variable region may comprise an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the light chain variable region of the same antibody clone of Table 1. The heavy chain variable region may comprise the amino acid sequence of a heavy chain variable region of an antibody clone of Table 1 and / or the light chain variable region may comprise the amino acid sequence of the same antibody clone of Table 1. The antibody or antigen binding fragment may comprise a heavy chain framework region (HFR) 1, HFR2, HFR3, and HFR4 and light chain framework region (LFR) 1, LFR2, LFR3, and LFR4, and wherein the HFR1, HFR2, HFR3, and HFR4 may comprise an amino acid sequence with at least 80% sequence identity to an HFR1, HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1, and the LFR1, LFR2, LFR3, and LFR4 comprises an amino acid sequence with at least 80% sequence identity to the LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone of Table 1. The antibody or antigen binding fragment may comprise aheavy chain framework region (HFR) 1, HFR2, HFR3, and HFR4 and light chain framework region (LFR) 1, LFR2, LFR3, and LFR4, and wherein the HFR1, HFR2, HFR3, and HFR4 may comprise an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to an HFR1, HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1, and the LFR1, LFR2, LFR3, and LFR4 comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone of Table 1. The HFR1, HFR2, HFR3, and HFR4 may comprise the amino acid sequence of an HFR1, HFR2, HFR3, and HFR4, respectively, of an antibody clone of Table 1, and the LFR1, LFR2, LFR3, and LFR4 may comprise the amino acid sequence of the LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone of Table 1. The antibody or antigen binding fragment may comprise a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence with at least 70% sequence identity to a heavy chain of an antibody clone of Table 1 and the light chain comprises an amino acid sequence with at least 70% sequence identity to the light chain of the same antibody clone of Table 1. The antibody or antigen binding fragment may comprise a heavy chain and a light chain and wherein the heavy chain comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to a heavy chain of an antibody clone of Table 1 and the light chain comprises an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the light chain of the same antibody clone of Table 1. The antibody or antigen binding fragment may comprise a heavy chain and a light chain and wherein the heavy chain comprises the amino acid sequence of an antibody clone of Table 1 and the light chain comprises the amino acid sequence of the same antibody clone of Table 1.

[0018] The heavy chain variable region may comprise a heavy chain framework region that has or has at least 80% sequence identity to a heavy chain framework region of an antibody clone of Table 1 and the light chain variable region comprises a light chain framework region that has or has at least 80% sequence identity to a light chain framework region of the sameantibody clone of Table 1. The heavy chain variable region may comprise a heavy chain framework region having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to a heavy chain framework region of an antibody clone of Table 1 and the light chain variable region comprises a light chain framework region having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to a light chain framework region of the same antibody clone of Table 1.

[0019] The heavy chain variable region may comprise at least 70% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 70% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 75% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 75% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 80% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 80% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 85% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 85% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 90% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 90% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs andthree light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 95% sequence identity to the heavy chain variable region of an antibody clone of Table 1 and the light chain variable region comprises at least 95% sequence identity to the light chain variable region of the same antibody clone of Table 1, and wherein the heavy chain and light chain comprise 100% sequence identity to each of the three heavy chain CDRs and three light chain CDRs from the same antibody clone of Table 1.

[0020] The antigen binding fragment may be at least 2, 3, 4, 5, or 6 scFv, F(ab’)2, Fab’, Fab, Fv, or rlgG, or combinations thereof. The polypeptide and / or antigen binding fragments of the disclosure may comprise a linker between a heavy chain and light chain variable region or between antigen binding fragments. The linker may be a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (G4S)n and (GGGS)n, where n is an integer of at least one. n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art and may be used as a linker in the polypeptides of the disclosure. Exemplary linkers can comprise or consist of GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, and the like.

[0021] The antibody or antigen binding fragment of the disclosure may be human, chimeric, or humanized. The antibody, or antigen binding fragment may bind a gamma delta TCR (including the Vy9V52 TCR) with a KD of about 10'6M / L or M to about 10'12M / L or M. The antibody or antigen binding fragment may bind TCR (including the Vy9V52 TCR) with a KD of about, a KD of at least, or a KD of at most 10, 10’1, 10'2, 10'3, 10'4, 10'5, 10'6, 10'7, 10'8, 10'9, 10'10, 10’11, IO’12, 10'13, 10'14, 10'15, 10'16, IO’17, or 10'18(or any derivable range therein) M, M / L, pM, nM, or pM. The antibody or antigen binding fragment may bind a gamma delta TCR (including the VY9V52 TCR) with a KD of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, or any range or value derivable therein. The antibody or antigen binding fragment may bind a gamma delta TCR (including the Vy9V52 TCR) with a KD of less than 2 nM. The antibody or antigen binding fragment may bind a gamma delta TCR (including the Vy9V52 TCR) with a KD of less than 1 nM. The antibody or antigen binding fragment may bind a gamma delta TCR (including the Vy9V52 TCR) with a KD of less than 0.5 nM. The antibody or antigen binding fragment may be further defined as a human antibody or antigen binding fragment, humanized antibody or antigen binding fragment, recombinant antibody or antigen binding fragment, chimeric antibody or antigen binding fragment, an antibody or antigen binding fragment derivative, a veneered antibody or antigen binding fragment, a diabody, a monoclonal antibody or antigen binding fragment, a single domainantibody, or a single chain antibody. The antigen binding fragment may be further defined as a single chain variable fragment (scFv), F(ab’)2, Fab’, Fab, Fv, or rlgG. The antibody, antigen binding fragment, or polypeptide may be operatively linked to a detectable label. Detectable labels are described herein.

[0022] Also described herein are multi-specific antibodies and polypeptides. Accordingly, bivalent or bispecific antibodies that comprise two antigen binding fragments, wherein the antigen binding fragment is two of the same antigen binding fragments or two different antigen binding fragments are provided by the description. The disclosure also provides for multispecific polypeptides. Also described are polypeptides comprising at least 2, 3, 4, 5, or 6 antigen binding fragments. The antigen binding fragment may be at least 2, 3, r, 5, or 6 scFv, F(ab’)2, Fab’, Fab, Fv, or rlgG, or combinations thereof.

[0023] Compositions may comprise more than one antibody and / or antigen binding fragment of the disclosure. Accordingly, compositions of the disclosure may comprise, may comprise at least, or may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antibodies and / or antigen binding fragments of the disclosure.

[0024] In aspects of the disclosure, it is also contemplated that 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, or 30 polypeptides of Table 1 or Table 2 may be excluded.

[0025] The patient may be further defined as a human patient or a mammalian patient. The patient may be a laboratory or veterinary animal, such as a pig, horse, cat, cow, rabbit, mouse, rat, or dog. The patient may have one or more symptom of cancer. The patient may not have any symptoms of cancer. The patient may be one that has been diagnosed with cancer. The patient may be one that has not been diagnosed with cancer. The patient may be one that has been previously treated for cancer. The patient may be one that has one or more symptoms of an infection. The patient may be one that does not have one or more symptoms of an infection. The patient may be one that has been diagnosed with an infection. The patient may be one that has not been diagnosed with an infection. The patient may be one that has symptoms of a disease treated with an immunotherapy. The patient may be one that does not have symptoms of a disease treated with an immunotherapy. The patient may be one that has been diagnosed with a disease indicated for an immunotherapy. The patient may be one that has not been diagnosed with a disease indicated for an immunotherapy.

[0026] The patient may be administered an additional therapy. The patient may be one that is resistant or has been determined to be resistant to the previous therapy. The additional therapy may comprise one or more of radiotherapy, chemotherapy, and immunotherapy.

[0027] The method may further comprise incubating the antibody, antigen binding fragment, or polypeptide under conditions that allow for the binding of the antibody, antigen binding fragment, or polypeptide to antigens in the biological sample or extract thereof. The method may further comprise detecting the binding of an antigen to the antibody, antigen binding fragment, or polypeptide. The method may further comprise contacting the biological sample with at least one capture antibody, antigen, or polypeptide. The at least one capture antibody, antigen binding fragment, or polypeptide may be an antibody, polypeptide, or antigen binding fragment of the disclosure. The capture antibody may be linked or operatively linked to a solid support. The term “operatively linked” refers to a situation where two components are combined or capable of combining to form a complex. For example, the components may be covalently attached and / or on the same polypeptide, such as in a fusion protein or the components may have a certain degree of binding affinity for each other, such as a binding affinity that occurs through van der Waals forces. The biological sample may comprise a blood sample, urine sample, fecal sample, or nasopharyngeal sample. The at least one antibody, antigen binding fragment, or polypeptide may be operatively linked to a detectable label. The method may further comprise incubating the antibody, antigen binding fragment, or polypeptide under conditions that allow for the binding of the antibody, antigen binding fragment, or polypeptide to antigens in the biological sample or extract thereof. The method may further comprise detecting the binding of an antigen to the antibody, antigen binding fragment, or polypeptide. The method may further comprise contacting the biological sample with at least one capture antibody, antigen, or polypeptide. The biological sample may comprise a blood sample, urine sample, fecal sample, or nasopharyngeal sample.

[0028] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of clone Tl, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of clone Tl, respectively.

[0029] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of clone T2, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of clone T2, respectively.

[0030] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a lightchain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of clone T3, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of clone T3, respectively.

[0031] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of clone T4, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of clone T4, respectively.

[0032] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of clone T5, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of clone T5, respectively.

[0033] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of clone T6, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of clone T6, respectively.

[0034] The disclosure describes an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region and a light chain variable region of any one of clones Tl, T2, T3, T4, T5, T6, T7, T7.2, T7.3, T7.4, T7.5, or T7.6.

[0035] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 1, 2, and 3, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 6, respectively.

[0036] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 13, and 14, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 15, respectively.

[0037] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:21, 22, and 23, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 24, respectively.

[0038] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:30, 31, and 32, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 33, respectively.

[0039] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:39, 40, and 41, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 42, respectively.

[0040] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS:48, 49, and 50, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 51, respectively.

[0041] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 57, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0042] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 57,respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:64, 5, and 58, respectively.

[0043] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 67, and 57, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0044] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 70, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0045] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 73, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0046] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region having a HCDR1, HCDR2, and HCDR3, and a light chain variable region having a LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 70, respectively and the LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence of SEQ ID NOS:64, 5, and 58, respectively.

[0047] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region and a light chain variable region of SEQ ID NOS:7 and 8, SEQ ID NOS: 16 and 17, SEQ ID NOS:25 and 26, SEQ ID NOS:34 and 35, SEQ ID NOS:43 and 44, SEQ ID NOS:52 and 53, SEQ ID NOS:59 and 60, SEQ ID NOS:59 and 65, SEQ ID NOS:68 and 60, SEQ ID NOS:71 and 60, SEQ ID NOS:74 and 60, or SEQ ID NOS:71 and 65.

[0048] Aspects of the disclosure relate to a fragment antibody or polypeptide comprising a heavy chain variable region of SEQ ID NOS:9, 18, 27, 36, 45, 54, or 61.

[0049] Aspects of the disclosure relate to an antibody, antigen binding fragment, or polypeptide comprising a heavy chain variable region and a light chain variable region of SEQ ID NOS: 10 and 11, SEQ ID NOS: 19 and 20, SEQ ID NOS:28 and 29, SEQ ID NOS:37 and 38, SEQ ID NOS :46 and 47, SEQ ID NOS:55 and 56, SEQ ID NOS: 62 and 63, SEQ ID NOS:61 and 66, SEQ ID NOS:69 and 62, SEQ ID NOS:72 and 62, SEQ ID NOS:75 and 62, or SEQ ID NOS:72 and 66.

[0050] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain variable region having a target HCDR1, target HCDR2, and target HCDR3, and a target light chain variable region having a target LCDR1, target LCDR2, and target LCDR3, wherein the target HCDR1, target HCDR2, and target HCDR3 comprises an amino acid sequence of SEQ ID NOS:76, 77, and 78, respectively and the target LCDR1, target LCDR2, and target LCDR3 comprises an amino acid sequence of SEQ ID NOS:79, 80, and 81, respectively, and a T7 heavy chain variable region having a T7 HCDR1, T7 HCDR2, and T7 HCDR3, and a T7 light chain variable region having a T7 LCDR1, T7 LCDR2, and T7 LCDR3, wherein the T7 HCDR1, T7 HCDR2, and T7 HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 57, respectively and the T7 LCDR1, T7 LCDR2, and T7 LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0051] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain variable region having a target HCDR1, target HCDR2, and target HCDR3, and a target light chain variable region having a target LCDR1, target LCDR2, and target LCDR3, wherein the target HCDR1, target HCDR2, and target HCDR3 comprises an amino acid sequence of SEQ ID NOS: 87, 88, and 89, respectively and the target LCDR1, target LCDR2, and target LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 90, respectively, and a T7 heavy chain variable region having a T7 HCDR1, T7 HCDR2, and T7 HCDR3, and a T7 light chain variable region having a T7 LCDR1, T7 LCDR2, and T7 LCDR3, wherein the T7 HCDR1, T7 HCDR2, and T7 HCDR3 comprises an amino acid sequence of SEQ ID NOS: 12, 22, and 57, respectively and the T7 LCDR1, T7 LCDR2, and T7 LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0052] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain variable region having a target HCDR1, target HCDR2, and target HCDR3, and a target light chain variable region having a target LCDR1, target LCDR2, and target LCDR3, wherein the target HCDR1, target HCDR2, and target HCDR3 comprises an amino acid sequence of SEQ ID NOS:94, 95, and 96, respectively and the target LCDR1, target LCDR2, and target LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 97, respectively, and aT7 light chain variable region having a T7 LCDR1, T7 LCDR2, and T7 LCDR3, wherein the T7 LCDR1, T7 LCDR2, and T7 LCDR3 comprises an amino acid sequence of SEQ ID NOS:4, 5, and 58, respectively.

[0053] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain scFv comprising an amino acid sequence of SEQ ID NO: 82, a target light chain scFv comprising an amino acid sequence of SEQ ID NO: 83, a T7 heavy chain scFv comprising an amino acid sequence of SEQ ID NO: 84, and a T7 light chain scFv comprising an amino acid sequence of SEQ ID NO: 85.

[0054] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain scFv comprising an amino acid sequence of SEQ ID NO: 91, a target light chain scFv comprising an amino acid sequence of SEQ ID NO: 92, a T7 heavy chain scFv comprising an amino acid sequence of SEQ ID NO: 84, and a T7 light chain scFv comprising an amino acid sequence of SEQ ID NO: 85.

[0055] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain comprising an amino acid sequence of SEQ ID NO: 98, a target light chain comprising an amino acid sequence of SEQ ID NO: 99, a T7 heavy chain scFv comprising an amino acid sequence of SEQ ID NO: 84, and a T7 light chain scFv comprising an amino acid sequence of SEQ ID NO: 85.

[0056] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target Fab heavy chain comprising an amino acid sequence of SEQ ID NO: 100, a target full light chain comprising an amino acid sequence of SEQ ID NO: 101, a target full heavy chain comprising an amino acid sequence of SEQ ID NO: 102, and a T7 scFv-Fc comprising an amino acid sequence of SEQ ID NO: 103.

[0057] Aspects of the disclosure relate to a BiTE or polypeptide comprising a target heavy chain scFv, a target light chain scFv, and a T7 heavy chain variable region and a T7 light chain variable region of SEQ ID NOS:7 and 8, SEQ ID NOS: 16 and 17, SEQ ID NOS:25 and 26, SEQ ID NOS:34 and 35, SEQ ID NOS:43 and 44, SEQ ID NOS:52 and 53, SEQ ID NOS:59 and 60, SEQ ID NOS:59 and 65, SEQ ID NOS:68 and 60, SEQ ID NOS:71 and 60, SEQ ID NOS:74 and 60, or SEQ ID NOS:71 and 65.

[0058] Aspects of the disclosure relate to a BiTE or polypeptide comprising an amino acid sequence of SEQ ID NO: 86 or 93.

[0059] Also disclosed are any one of the following aspects:

[0060] Aspect 1 includes an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable regioncomprises HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, and HCDR3 from a heavy chain variable region of an antibody clone of Table 1, and wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

[0061] Aspect 2 depends upon Aspect 1, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence of HCDR1, HCDR2, and HCDR3 of a clone of Table 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequence of LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1.

[0062] Aspect 3 depends upon Aspect 1 or 2, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence that has at least 80% sequence identity to the corresponding CDRs of Table 1, from the same antibody clone.

[0063] Aspect 4 depends upon Aspect 1 or 2, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequence of the corresponding CDRs of Table 1, from the same antibody clone.

[0064] Aspect 5 depends upon any one of Aspects 1 to 4, wherein the heavy chain variable region comprises an amino acid sequence with at least 80% sequence identity to a heavy chain variable region (VH) of an antibody clone of Table 1 and / or the light chain variable region comprises an amino acid sequence with at least 80% sequence identity to the light chain variable region (VL) of the same antibody clone of Table 1.

[0065] Aspect 6 depends upon Aspect 5, wherein the heavy chain variable region comprises the amino acid sequence of a heavy chain variable region of an antibody clone of Table 1 and / or the light chain variable region comprises the amino acid sequence of the same antibody clone of Table 1.

[0066] Aspect 7 depends upon any one of Aspects 1 to 6, wherein the antibody or antigenbinding fragment comprises a full heavy chain with at least 80% sequence identity to a full heavy chain of an antibody clone of Table 1, and comprises a full light chain with at least 80% sequence identity to a full light chain of the same antibody clone of Table 1.

[0067] Aspect 8 depends upon any one of Aspects 1 to 6, wherein the antibody or antigenbinding fragment comprises a full heavy chain of an antibody clone of Table 1, and comprises a full light chain of the same antibody clone of Table 1.

[0068] Aspect 9 depends upon any one of Aspects 1 to 8, wherein the antibody comprises a heavy chain Fab with at least 80% sequence identity to a full heavy chain Fab of an antibody clone of Table 1.

[0069] Aspect 10 depends upon Aspect 9, wherein the heavy chain Fab comprises a full heavy chain Fab of an antibody clone of Table 1.

[0070] Aspect 11 includes an antibody or antigen-binding fragment comprising a variable region capable of binding a public domain of a Vy9V52 T-cell receptor protein.

[0071] Aspect 12 depends upon Aspect 11, wherein the variable region comprises one, two, three, four, five, or six CDRs capable of binding the public domain of the Vy9V52 T-cell receptor protein.

[0072] Aspect 13 depends upon Aspect 12, wherein one, two, three, four, five, or six of the CDRs comprise an amino acid sequence of VXY, where X is any amino acid.

[0073] Aspect 14 depends upon Aspect 11, wherein the antigen or antigen-binding fragment comprises HCDR3 of clone T7 in Table 1.

[0074] Aspect 15 includes an antibody or antigen-binding fragment capable of binding a public domain of a Vy9V52 T-cell receptor protein, wherein the antibody or antigen-binding fragment comprises a CDR capable of binding to amino acids TLG located in the public domain of the Vy9V52 T-cell receptor protein, and wherein the CDR comprises an amino acid sequence of VXY, where X is any amino acid.

[0075] Aspect 16 includes an antibody or antigen-binding fragment capable of binding a public domain of a Vy9V52 T-cell receptor protein, wherein the antibody or antigen-binding fragment comprises a CDR capable of binding to the main chain of the public domain located in the public domain of the Vy9V52 T-cell receptor protein.

[0076] Aspect 17 depends upon any one of Aspects 1 to 16, wherein the antibody is human, chimeric, or humanized.

[0077] Aspect 18 depends upon any one of Aspects 1 to 17, wherein the antibody or antigen-binding fragment binds a Vy9V52 T-cell receptor protein with a KD of about 10A-6 M / L to about 10A-12 M / L.

[0078] Aspect 19 depends upon any one of Aspects 1 to 18, wherein the antibody is a neutralizing antibody.

[0079] Aspect 20 depends upon any one of Aspects 1 to 19, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody.

[0080] Aspect 21 depends upon any one of Aspects 1 to 19, wherein the antigen-binding fragment is a single chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv, or rlgG.

[0081] Aspect 22 includes a bi-specific antibody or antigen-binding fragment comprising two distinct variable regions having a light chain variable region and a heavy chain variable region, wherein one of the two distinct variable regions comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, and HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

[0082] Aspect 23 depends upon Aspect 22, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the one variable region has at least 95% sequence identity to the HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of the antibody clone of Table 1, and wherein the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the one variable region has at least 95% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

[0083] Aspect 24 depends upon Aspect 22, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the one variable region has at least 100% sequence identity to the HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of the antibody clone of Table 1, and wherein the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the one variable region has at least 100% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

[0084] Aspect 25 depends upon Aspect 22, wherein the heavy chain variable region of the one variable region comprises HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to SEQ ID NOs: 12, 22, and 57, respectively, and wherein the light chain variable region of the one variable region comprises LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to SEQ ID NOs:4, 5, and 58, respectively.

[0085] Aspect 26 depends upon Aspect 22, wherein the heavy chain variable region of the one variable region comprises HCDR1, HCDR2, and HCDR3 having at least 95% sequence identity to SEQ ID NOs: 12, 22, and 57, respectively, and wherein the light chain variable region of the one variable region comprises LCDR1, LCDR2, and LCDR3 having at least 95% sequence identity to SEQ ID NOs:4, 5, and 58, respectively.

[0086] Aspect 27 depends upon Aspect 22, wherein the heavy chain variable region of the one variable region comprises HCDR1, HCDR2, and HCDR3 comprising SEQ ID NOs: 12, 22,and 57, respectively, and wherein the light chain variable region of the one variable region comprises LCDR1, LCDR2, and LCDR3 comprising SEQ ID NOs:4, 5, and 58, respectively.

[0087] Aspect 28 depends upon any one of Aspects 22 to 27, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region that are able to bind to a tumor cell.

[0088] Aspect 29 depends upon any one of Aspects 22 to 28, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region that are able to bind to an analyte.

[0089] Aspect 30 depends upon any one of Aspects 22 to 29, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region that are able to bind to a tumor antigen.

[0090] Aspect 31 depends upon any one of Aspects 22 to 30, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region from atezolizumab, belantamab, bevacizumab, blinatumomab, brentuximab, cetuximab, daratumumab, dinutuximab, elotuzumab, enfortumab, gemtuzumab, ibritumomab, inotuzumab, necitumumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pertuzumab, ramucirumab, rituximab, sacituzumab, or trastuzumab.

[0091] Aspect 32 depends upon any one of Aspects 22 to 31, wherein the antibody is human, chimeric, or humanized.

[0092] Aspect 33 depends upon any one of Aspects 22 to 32, wherein the antibody or antigen-binding fragment binds a Vy9V52 T-cell receptor protein with a KD of about 10'6M / L to about IO’12M / L.

[0093] Aspect 34 depends upon any one of Aspects 22 to 33, wherein the antibody is a bispecific T cell engager antibody.

[0094] Aspect 35 depends upon any one of Aspects 22 to 34, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, or a monoclonal antibody.

[0095] Aspect 36 includes a polypeptide comprising the antigen-binding fragment of any one of Aspects 1 to 35.

[0096] Aspect 37 depends upon Aspect 36, wherein the polypeptide comprises at least two antigen-binding fragments, wherein each antigen-binding fragment is independently selected from an antigen-binding fragment of any one of Aspects 1 to 21.

[0097] Aspect 38 depends upon Aspect 36 or 37, wherein the polypeptide is multivalent.

[0098] Aspect 39 depends upon any one of Aspects 36 to 38, wherein the polypeptide is bispecific.

[0099] Aspect 40 includes a composition comprising the antibody or antigen-binding fragment of any one of Aspects 1 to 35 or the polypeptide of any one of Aspects 36 to 39.

[0100] Aspect 41 depends upon Aspect 40, wherein the composition comprises a pharmaceutical excipient.

[0101] Aspect 42 depends upon Aspect 40 or 41, wherein the composition further comprises an adjuvant.

[0102] Aspect 43 depends upon any one of Aspects 40 to 42, wherein the composition is formulated for parenteral, intravenous, subcutaneous, intramuscular, or intranasal administration.

[0103] Aspect 44 depends upon any one of Aspects 1 to 43, wherein the composition comprises at least two antibodies or antigen-binding fragments.

[0104] Aspect 45 includes one or more nucleic acids encoding the antibody or antigenbinding fragment of any one of Aspects 1 to 20 or the polypeptide of Aspect 39.

[0105] Aspect 46 includes a vector comprising the nucleic acid(s) of Aspect 45.

[0106] Aspect 47 includes a host cell comprising the nucleic acid of Aspect 45, or the vector of Aspect 46.

[0107] Aspect 48 depends upon Aspect 47, wherein the host cell is a human cell, B cell, T cell, Chinese hamster ovary, NS0 murine myeloma cell, or PER.C6 cell.

[0108] Aspect 49 includes a method of making a cell comprising transferring the nucleic acid(s) of Aspect 45 or the vector of Aspect 46 into a cell.

[0109] Aspect 50 depends upon Aspect 49, wherein the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.

[0110] Aspect 51 depends upon Aspect 50, wherein the method further comprises isolating the expressed polypeptide.

[0111] Aspect 52 depends upon any one of Aspects 49 to 51, wherein the cell is a human cell, B cell, T cell, Chinese hamster ovary, NS0 murine myeloma cell, or PER.C6 cell.

[0112] Aspect 53 includes a method for producing a polypeptide comprising transferring the nucleic acid(s) of Aspect 45 or the vector of Aspect 46 into a cell and isolating polypeptides expressed from the nucleic acid.

[0113] Aspect 54 depends upon Aspect 53, wherein the cell is a human cell, B cell, T cell, Chinese hamster ovary, NS0 murine myeloma cell, or PER.C6 cell.

[0114] Aspect 55 includes a method for treating or preventing cancer in a patient, the method comprising administering to the patient the antibody or antigen-binding fragment of any one of Aspects 1 to 35, the polypeptide of any one of Aspects 36 to 39, the composition of any one of Aspects 40 to 44, or the host cell of Aspect 47 or 48.

[0115] Aspect 56 depends upon Aspect 55, wherein the patient is a human patient.

[0116] Aspect 57 depends upon Aspect 55 or 56, wherein the patient has one or more symptoms of cancer.

[0117] Aspect 58 depends upon Aspect 55 or 56, wherein the patient does not have any symptoms of cancer.

[0118] Aspect 59 depends upon any one of Aspects 55 to 58, wherein the patient has been diagnosed with cancer.

[0119] Aspect 60 depends upon any one of Aspects 55 to 58, wherein the patient has not been diagnosed with cancer.

[0120] Aspect 61 depends upon any one of Aspects 55 to 60, wherein the patient has been previously treated for cancer.

[0121] Aspect 62 depends upon any one of Aspects 55 to 61, wherein the patient is administered an additional therapy.

[0122] Aspect 63 depends upon Aspect 62, wherein the additional therapy comprises radiotherapy, chemotherapy, or immunotherapy.

[0123] Aspect 64 includes a method for treating or preventing a disease indicated for an immunotherapy in a patient, the method comprising administering to the patient the antibody or antigen-binding fragment of any one of Aspects 1 to 35, the polypeptide of any one of Aspects 36 to 39, the composition of any one of Aspects 40 to 44, or the host cell of Aspect 47 or 48.

[0124] Aspect 65 depends upon Aspect 64, wherein the patient is a human patient.

[0125] Aspect 66 depends upon Aspect 64 or 65, wherein the patient has one or more symptoms of the disease.

[0126] Aspect 67 depends upon Aspect 64 or 65, wherein the patient does not have one or more symptoms of the disease.

[0127] Aspect 68 depends upon Aspect 64 or 65, wherein the patient has been diagnosed with the disease.

[0128] Aspect 69 depends upon Aspect 64 or 65, wherein the patient has not been diagnosed with the disease.

[0129] Aspect 70 depends upon any one of Aspects 64 to 69, wherein the patient has been previously treated for the disease.

[0130] Aspect 71 depends upon Aspect 70, wherein the patient was resistant to the previous treatment.

[0131] Aspect 72 includes a method for evaluating a sample from a patient, the method comprising contacting a biological sample from the patient, or extract thereof, with at least one antibody, antigen-binding fragment, or polypeptide of any one of Aspects 1 to 39.

[0132] Aspect 73 depends upon Aspect 72, wherein the at least one antibody, antigenbinding fragment, or polypeptide is operatively linked to a detectable label.

[0133] Aspect 74 depends upon Aspect 72 or 73, wherein the method further comprises incubating the antibody, antigen-binding fragment, or polypeptide under conditions that allow for the binding of the antibody, antigen-binding fragment, or polypeptide to antigens in the biological sample or extract thereof.

[0134] Aspect 75 depends upon any one of Aspects 72 to 74, wherein the method further comprises detecting the binding of an antigen to the antibody, antigen-binding fragment, or polypeptide.

[0135] Aspect 76 depends upon any one of Aspects 72 to 75, wherein the method further comprises contacting the biological sample with at least one capture antibody, antigen, or polypeptide.

[0136] Aspect 77 depends upon Aspect 76, wherein the at least one capture antibody, antigen-binding fragment, or polypeptide comprises at least one antibody or antigen-binding fragment of Aspects 1 to 20.

[0137] Aspect 78 depends upon Aspect 76 or 77, wherein the capture antibody or fragment is linked to a solid support.

[0138] Aspect 79 depends upon any one of Aspects 72 to 78, wherein the biological sample comprises a tissue sample or a blood sample.

[0139] Aspect 80 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T1 in Table 1.

[0140] Aspect 81 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T2 in Table 1.

[0141] Aspect 82 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T3 in Table 1.

[0142] Aspect 83 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T4 in Table 1.

[0143] Aspect 84 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T5 in Table 1.

[0144] Aspect 85 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T6 in Table 1.

[0145] Aspect 86 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7 in Table 1.

[0146] Aspect 87 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.2 in Table 1.

[0147] Aspect 88 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.3 in Table 1.

[0148] Aspect 89 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.4 in Table 1.

[0149] Aspect 90 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.5 in Table 1.

[0150] Aspect 91 includes an antibody or antigen-binding fragment comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.6 in Table 1.

[0151] Aspect 92 includes an antibody or antigen-binding fragment comprising SEQ ID NOs:82-85.

[0152] Aspect 93 includes an antibody or antigen-binding fragment comprising SEQ ID NOs:84, 85, 90, and 91.

[0153] Aspect 94 includes an antibody or antigen-binding fragment comprising SEQ ID NOs:84, 85, 98, and 99.

[0154] Aspect 95 includes a method of treating a patient, the method comprising administering the antibody of any one of Aspects 80 to 94 to the patient.

[0155] Aspect 96 includes a method of treating a patient, the method comprising administering a pharmaceutical composition comprising the antibody of any one of Aspects 80 to 94 to the patient.

[0156] Aspect 97 includes a method of engaging a T cell with a cancer cell, the method comprising contacting the T cell and the cancer cell with the antibody or antigen-binding fragment of any one of Aspects 22 to 35.

[0157] Aspect 98 depends upon Aspect 97, wherein the T cell is a y952 T cell.

[0158] Aspect 99 depends upon Aspect 97 or 98, wherein the antibody or antigen-binding fragment comprises a variable region that is able to bind to an antigen expressed on the cancer cell.

[0159] Aspect 100 depends upon Aspect 99, wherein the antigen expressed on the cancer cell is a tumor antigen.

[0160] Aspect 101 depends upon any one of Aspects 97 to 100, wherein the contacting occurs in vivo.

[0161] Aspect 102 depends upon any one of Aspects 97 to 100, wherein the contacting occurs ex vivo.

[0162] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0163] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0164] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0165] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0166] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention. As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that embodiments or aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0167] “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.

[0168] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the disclosure may apply to any other embodiment or aspect of the disclosure. Furthermore, any composition of the disclosure may be used in any method of the disclosure, and any method of the disclosure may be used to produce or to utilize any composition of the disclosure. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0169] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0170] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0171] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0172] FIGs. 1A-1G: Generating sABs against Vy9V<52 T-cells.(A) The schematic depicts the Fab-fused phage display biopanning process and the subsequent conversion of the selected synthetic antibodies (sABs) into a bispecific T-cell engager (BiTE) format. This BiTE is designed to activate Vy9V52 T cells and induce cancer cell killing. (B) The phage ELISA results show the binding profiles of the selected sABs. (C) The multi-pointELISA data demonstrate the binding specificity of the lead candidate Fab T7 against the Vy9V52 T cell receptor (TCR) (blue), Vy4V52 TCR (green), or Vy9V51 TCR (red). The results indicate a strong preference for the Vy9V52 TCR, although the binding is primarily driven by the interaction with the V52 chain (n = 3, mean ± SD). (D) The size exclusion chromatography profile shows the increased size of the T7-Vy9V52 TCR complex compared to the Vy9V52 TCR alone, indicating the formation of a stable complex. (E) The thermal melting curves, as monitored by differential scanning fluorimetry (DSF), demonstrate that the stability of the Vy9V52 TCR increased by 1°C when it formed a complex with Fab T7 (n = 3). (F) The surface plasmon resonance (SPR) sensogram shows the kinetics of binding between Fab T7 and the Vy9V52 TCR, characterized by a slow dissociation rate. (G) The SPR-derived binding kinetics data for all the selected sABs and the Vy9V52 TCR are presented.

[0173] FIGs. 2A-2E: Structural insights of T7 Fab engaged with the Vy9V<52 TCR. (A) The crystal structure of Fab T7 bound to the G115 Vy9V52 T cell receptor (TCR) (PDB: XXXX) is shown. (B) The surface representation depicts the interaction between Fab T7 and the Gi l 5 Vy9V52 TCR. Amino acid residues from the Fab complementarity-determining region (CDR) loops that are involved in an extensive network of 19 hydrogen bonds with the Vy9V52 TCR are displayed as sticks. (C) Key residues from the Vy9V52 TCR that participate in the interaction with Fab T7 are shown. The orientation of the Fab T7 CDR loops is represented as a cartoon. (D) Fab T7 forms three hydrogen bonds with the public motif (TLG) in the Vy9V52 CDR3. The main chain of Vall03 from the Fab CDR-H3 forms two hydrogen bonds with the nitrogen from the main chain of Leu97 and Gly98. These interactions contribute to the pan-specific binding characteristics of Fab T7 to a diverse portfolio of Vy9V52 TCRs. The CDR3 from Vy9V52 TCR is shown as cartoon. (E) The surface representation of the Vy9V52 TCR highlights the interactions with Fab T7 (green) and BTN2A1 (red), suggesting the possibility of simultaneous interaction of the Vy9V52 TCR with both molecules.

[0174] FIGs. 3A-3H: Pan-specific T7 Fab binds and activates Vy9V62 T-cells. (A) The binding of Vy9V52 T cell receptor (TCR) Fabs to G115-TCR expressing Jurkat Jrt-3.3 cells was assessed using flow cytometry (n = 3, mean ± SD). (B) The T7 Fab binds to peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. (C) The universality of the T7 antibody was tested by evaluating its binding to PBMCs isolated from twenty healthy donors. (D) The T7 Fab efficiently activates T cells, as demonstrated by the increased CD69 expression on PBMCs isolated from healthy donors (n = 3, mean ± SD). (E) The WebLogo analysis shows the amino acid diversity in the complementarity-determining region 3 (CDR3) of the V52 TCR. The black box indicates the public motif (TLG) at positions 96-98, commonlyfound in the sequenced Vy9V52 T cells, and recognized by the T7 antibody, as shown by the crystal structure. (F) The pan-specificity of the T7 Fab was tested by introducing mutations in the CDR3 of the Vy9V52 TCR at positions 96-98. (G) Binding of T7 IgG is exclusive to Vy9V52 T cells, with no detectable binding to other T cell populations. Representative histograms for the isotype control IgG (negative control) are displayed in the upper panels, while histograms for T7 IgG staining are shown in the lower panels. A pronounced rightward shift in the histogram is observed exclusively for T7 IgG binding to Vy9V52 T cells, indicating robust and selective recognition. (H) Binding of T7 IgG to Vy9V52 T cells, assessed by flow cytometry over a concentration range, with EC50 = 0.19 nM (n=3, mean ± SD).

[0175] FIGs. 4A-4C: Efficient Stimulation of HCC-1954 Cancer Cell Cytotoxicity by T7 Fab via Vy9V62 T-cells. (A) Schematic representation of the plug-and-play BiTE platform. This modular system enables the coupling of two Fabs using the engineered protein GAI, which can be paired with either the human kappa Fab scaffold (FabH) or the synthetic LRT Fab scaffold (FabLRT). Notably, GAI exhibits ultra-high affinity for FabLRTbut does not bind to FabH. (B) Illustration of the cell killing assay. FabH, recognizing the Her2 extracellular domain on antigen-presenting cells (APCs), is linked to GAI via a 23-residue linker. Prior to the experiment, T7 FabLRTor UCHT1 FabLRTwere pre-complexed with HER2HGA1 to form a BiTE. To assess the BiTE's effect, 20,000 HCC-1954 cells were cultured overnight, followed by the addition of 200,000 PBMCs enriched for human gamma / delta T-cells (via alpha / delta TCR negative selection) and 50 nM of the BiTE construct. The cytotoxic effect was quantified by measuring the lactate dehydrogenase (LDH) activity released by dying cells after 24 hours of incubation. (C) Cytotoxicity profiles upon BiTE treatment. The T7 BiTE demonstrated efficient killing of HCC-1954 cells, comparable to the CD3-targeting UCHT1 BiTE. As a control, individual components of the plug-and-play BiTEs were tested, and none of them induced significant HCC-1954 cancer cell killing.

[0176] FIGs. 5A-5B: Bispecific T7 BiTE mediates lysis of PC3 cancer cells. (A) PC3 cancer cells were co-cultured with peripheral blood mononuclear cells (PBMCs) at a target-to- effector (T:E) ratio of 1 : 10 and treated with increasing concentrations of Her2-T7 BiTE, Her2- Okt3 BiTE, or RBD1-T7 BiTE (negative control). Cytotoxicity was assessed by quantifying lactate dehydrogenase (LDH) release from lysed cells following 48 hours of incubation. Data are presented as mean ± SEM (n = 4). (B) PC3 cancer cells were co-cultured with Vy9V52 T cells, enriched from PBMCs, at a T:E ratio of 1 :3 and treated with varying concentrations of Her2-T7 BiTE, Her2-Okt3 BiTE, or RBD1-T7 BiTE (negative control). Cytotoxicity wasdetermined by measuring LDH release after 48 hours of incubation. Data represent mean ± SEM (n = 4).

[0177] FIGs. 6A-6C: Cytokine release under Her2-T7 BiTE activation by Vy9V62 T cells. The measurement of IL-17 levels (pg / ml) in 48h co-culture of PC3 cells with (A) PBMC cells (1 : 10 T:E ratio) or (B) Vy9V52-enriched cells (1 :3 T :E ratio) over the concentration range of Her2-T7 BiTE, Her2-Okt3 BiTE, and RBD1-T7 BiTE (negative control). Vy9V52 T cells as opposite to a / 3 T cells do not release IL17. (C) IFNy levels (pg / ml) in 48h co-culture of PC3 cells with Vy9V52-enriched cells (1 :3 T:E ratio) over the concentration range of Her2-T7 BiTE, Her2 Okt3 BiTE. Data represent mean and SEM (n=3).

[0178] FIGs. 7A-7C: Bispecific T7 BiTE induces targeted lysis of PC3 cancer cells by Vy9V62 T cells, as visualized using the Incucyte S3 Imaging System. (A) Representative Incucyte images illustrate cancer cell lysis (right) in the presence of 1.56 nM Her2-T7 IgG, compared to isotype BiTE-treated controls (left). Clusters of lysed cells, indicative of cell death, are marked by arrows. (B, C) Quantitative cytotoxicity profiles following Her2-T7 BiTE treatment. Her2-T7 BiTE mediates potent and concentration-dependent killing of PC3 cells, as measured by changes in cell confluence using the Incucyte S3 Imaging System over a 70-hour co-culture period with Vy9V52-enriched T cells (target-to-effector ratio 1 :3).

[0179] FIG. 8: NT5E-T7 BiTE mediates lysis of PC3 cancer cells. PC3 cancer cells were co-cultured with Vy9V52 T cells, enriched from PBMCs, at a T:E ratio of 1 :3 and treated with varying concentrations of NT5E-T7 BiTE, or RBD1-T7 BiTE (negative control). Cytotoxicity was determined by measuring LDH release after 48 hours of incubation. Data represent mean ± SEM (n = 4).

[0180] FIG. 9: PRLR-targeting P2C2-T7 bi-IgG mediates lysis of MCF-7 cancer cells. MCF-7 cancer cells were co-cultured with Vy9V52 T cells, enriched from PBMCs, at a T:E ratio of 1:3 and treated with varying concentrations of P2C2-T7 bi-IgG, or RBD1-T7 bi-IgG (negative control). Cytotoxicity was determined by measuring LDH release after 48 hours of incubation. Data represent mean ± SEM (n = 4).

[0181] FIG. 10: Structure-guided design of T7 mutants for enhanced developability. ELISA results showing the binding profiles of T7 Fab mutants to Vy9V52TCR. Mutants T7.2 and T7.5 exhibited binding profiles indistinguishable from the wild-type, indicating that these substitutions do not perturb antigen recognition, while mutants T7.3, T7.4, and T7.6 demonstrated moderately reduced binding to Vy9V52TCR.

[0182] FIGs. 11A-11D: Comparative cytotoxicity of Her2-T7 BiTE and Her2-T7 bi- IgG against multiple cancer cell lines. PC3 (A), OVCAR-3 (B), HeLa (C), and PANCI (D)cancer cells were co-cultured with the expanded Vy9V52 T cells at a target-to-effector (T:E) ratio of 1 :3 and treated with increasing concentrations of Her2-T7 BiTE or Her2-T7 bi-IgG. Cytotoxicity was assessed by quantifying lactate dehydrogenase (LDH) release from lysed cells after 48 hours of incubation. Her2-OKT3 BiTE, targeting CD3, served as a positive control, while a BiTE targeting the SARS-CoV-2 RBD protein was used as an isotype control. Data are presented as mean ± SEM (n = 4).DETAILED DESCRIPTIONI. Antibodies

[0183] Aspects of the disclosure relate to antibodies, antigen binding fragments thereof, or polypeptides capable of specifically binding to a TCR.

[0184] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity.

[0185] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that are capable of interacting with different antibodies.

[0186] The term “epitope” includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T-cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0187] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques are well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockb erg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82: 178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0188] The term “immunogenic sequence” means a molecule that includes an amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term “immunogenic composition” means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0189] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human. The antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013).

[0190] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (K) and lambda ( ). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide.

[0191] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CHI, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonalantibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the aminoterminus of the polypeptide, and the CH domains are at the carboxy-terminus, with the CH3 being closest to the — COOH end. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (p), delta (5), gamma (y), alpha (a), or epsilon (a) chains, respectively. IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM subtypes include IgMl and IgM2. IgA subtypes include IgAl and IgA2.A. Types of Antibodies

[0192] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e.g., F(ab')2, Fab', Fab, Fv, and the like), including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins.

[0193] The term “monomer” means an antibody containing only one Ig unit. Monomers are the basic functional units of antibodies. The term “dimer” means an antibody containing two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc, or fragment crystallizable, region). The complex may be stabilized by a joining (J) chain protein. The term “multimer” means an antibody containing more than two Ig units attached to one another via constant domains of the antibody heavy chains (the Fc region). The complex may be stabilized by a joining (J) chain protein.

[0194] The term “bivalent antibody” means an antibody that comprises two antigenbinding sites. The two binding sites may have the same antigen specificities or they may be bispecific, meaning the two antigen-binding sites have different antigen specificities.

[0195] Bispecific antibodies are a class of antibodies that have two paratopes with different binding sites for two or more distinct epitopes. In some aspects, bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen. In some aspects, bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies”. Single domain antibodies are sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by alinker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No. W02010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which are specifically incorporated herein by reference in their entirety.

[0196] Bispecific antibodies can be constructed as: a whole IgG, Fab'2, Fab'PEG, a diabody, or alternatively as scFv. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti -idiotypic reaction. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148: 1547-1553 (1992), each of which are specifically incorporated by reference in their entirety.

[0197] In certain aspects, the antigen-binding domain may be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind a different antigen. For example, the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Accordingly, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells.

[0198] In some aspects, multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art. One such example is diabodies that are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites. The linker functionality is applicable for aspects of triabodies, tetrabodies, and higher order antibody multimers, (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2: 1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0199] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be advantageous because they can be readily constructed and expressed in E. coli. Diabodies (and other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94 / 13804) from libraries. If one arm of the diabody is kept constant, for instance, with a specificity directed against a protein, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described inRidgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39: 167-173, 2017), each of which is hereby incorporated by reference in their entirety.

[0200] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities. See, e.g., U.S. Patent No. 6,010,902, incorporated herein by reference in its entirety.

[0201] The part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope.” The paratope consists of the amino acid residues that make contact with the epitope of an antigen to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of the two variable domains, VH and VL, in dimerized configuration. The primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, Framework Regions (FR). The hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term “Complementarity Determining Region (CDR).” The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have high primary sequence similarity / consensus. The consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions. The hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur. CDRs in the VL domain are identified as LI, L2, and L3, with LI occurring at the most distal end and L3 occurring closest to the CL domain. The CDRs may also be given the names CDR-L1 (or LCDR1), CDR-L2 (or LCDR2), and CDR-L3 (or LCDR3). The L3 (CDR-L3) is generally the region of highest variability among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain arranged linearly in the primary structure, and separated from each other by Framework Regions. The amino terminal (N- terminal) end of the VL chain is named FR1. The region identified as FR2 occurs between LI and L2 hypervariable loops. FR3 occurs between L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which includes three CDRs identified as CDR-H1 (or HCDR1), CDR-H2 (or HCDR2), and CDR-H3 (or HCDR3). The majority of amino acid residues in the variable domains, or Fv fragments (VH and VL), are part of the framework regions (approximately 85%). The three dimensional, or tertiary, structure of an antibody molecule is such that the framework regionsare more internal to the molecule and provide the majority of the structure, with the CDRs on the external surface of the molecule.

[0202] Several methods have been developed and can be used by one skilled in the art to identify the exact amino acids that constitute each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms, which identify the conserved amino acid residues that make up the framework regions, therefore identifying the CDRs that may vary in length but are located between framework regions. Three commonly used methods have been developed for identification of the CDRs of antibodies: Kabat (as described in T. T. Wu and E. A. Kabat, “AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (as described in M.-P. Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). These methods each include unique numbering systems for the identification of the amino acid residues that constitute the variable regions. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen occur in the CDRs, although in some cases, residues within the framework regions contribute to antigen binding.

[0203] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include:

[0204] 1) Computational predictions of the tertiary structure of the antibody / epitope binding interactions based on the chemical nature of the amino acid sequence of the antibody variable region and composition of the epitope.

[0205] 2) Hydrogen-deuterium exchange and mass spectroscopy

[0206] 3) Polypeptide fragmentation and peptide mapping approaches in which one generates multiple overlapping peptide fragments from the full length of the polypeptide and evaluates the binding affinity of these peptides for the epitope.

[0207] 4) Antibody Phage Display Library analysis in which the antibody Fab fragment encoding genes of the mammal are expressed by bacteriophage in such a way as to be incorporated into the coat of the phage. This population of Fab expressing phage are then allowed to interact with the antigen which has been immobilized or may be expressed in by a different exogenous expression system. Non-binding Fab fragments are washed away, therebyleaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be readily isolated and the genes which encode them determined. This approach can also be used for smaller regions of the Fab fragment including Fv fragments or specific VH and VL domains as appropriate.

[0208] In certain aspects, affinity matured antibodies are enhanced with one or more modifications in one or more CDRs thereof that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s). Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and / or framework residues employed in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009) in conjugation with computation methods as demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).

[0209] Chimeric immunoglobulins are the products of fused genes derived from different species; “humanized” chimeras generally have the framework region (FR) from human immunoglobulins and one or more CDRs are from a non-human source.

[0210] In certain aspects, portions of the heavy and / or light chain are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851 (1984). For methods relating to chimeric antibodies, see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851- 6855 (1985), each of which are specifically incorporated herein by reference in their entirety. CDR grafting is described, for example, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are all hereby incorporated by reference for all purposes.

[0211] In some aspects, minimizing the antibody polypeptide sequence from the non- human species optimizes chimeric antibody function and reduces immunogenicity. Specific amino acid residues from non-antigen recognizing regions of the non-human antibody are modified to be homologous to corresponding residues in a human antibody or isotype. One example is the “CDR-grafted” antibody, in which an antibody comprises one or more CDRs from a particular species or belonging to a specific antibody class or subclass, while theremainder of the antibody chain(s) is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For use in humans, the V region composed of CDR1, CDR2, and partial CDR3 for both the light and heavy chain variance region from a non-human immunoglobulin, are grafted with a human antibody framework region, replacing the naturally occurring antigen receptors of the human antibody with the non-human CDRs. In some instances, corresponding non-human residues replace framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1 : 105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239: 1534-36 (1988).

[0212] Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0213] Polyclonal antibody preparations typically include different antibodies against different determinants (epitopes). In order to produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the antigen or antigen fragment, generally with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are subsequently collected from the sera of the host. The polyclonal antibody can be affinity purified against the antigen rendering it monospecific.

[0214] Monoclonal antibodies or “mAb” refer to an antibody obtained from a population of homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.B. Functional Antibody Fragments and Antigen-Binding Fragments1. Antigen-Binding Fragments

[0215] Certain aspects relate to antibody fragments, such as antibody fragments that bind to a TCR. The term functional antibody fragment includes antigen-binding fragments of an antibody that retain the ability to specifically bind to an antigen. These fragments are constituted of various arrangements of the variable region heavy chain (VH) and / or light chain(VL); and in some aspects, include constant region heavy chain 1 (CHI) and light chain (CL). In some aspects, they lack the Fc region constituted of heavy chain 2 (CH2) and 3 (CH3) domains. Aspects of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CHI domains; (ii) the Fd fragment type constituted with the VH and CHI domains; (iii) the Fv fragment type constituted with the VH and VL domains; (iv) the single domain fragment type, dAb, (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015), each of which are incorporated by reference.

[0216] Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 complementarity determining regions (CDRs) from a light chain variable region. Fusions of CDR-containing sequences to an Fc region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein.

[0217] The term Fab fragment (also “Fab” or “FAB”) means a monovalent antigen-binding fragment of an antibody containing the VL, VH, CL and CHI domains. The term Fab' fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes the VL, VH, CL and CHI domains and all or part of the hinge region. The term F(ab')2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab' fragments linked by a disulfide bridge at the hinge region. An F(ab')2 fragment includes, for example, all or part of the two VH and VL domains, and can further include all or part of the two CL and CHI domains.

[0218] The term Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs. An Fd fragment can further include CHI region sequences.

[0219] The term Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CHI domains. The VL and VH include, for example, the CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions have been connected by a flexiblelinker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference. The term (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain comprises self-associating a- helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as “miniantibodies” or “minibodies.”

[0220] A single domain antibody is an antigen-binding fragment containing only a VH or the VL domain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.2. Fragment Antigen Binding Region, Fab

[0221] Fab polypeptides of the disclosure include the Fab antigen binding fragment of an antibody. Unless specifically stated otherwise, the term “Fab” relates to a polypeptide excluding the Fc portion of the antibody. The Fab may be conjugated to a polypeptide comprising other components, such as further antigen binding domains, costimulatory domains, linkers, peptide spacers, transmembrane domains, endodomains, and accessory proteins. Fab polypeptides can be generated using conventional techniques known in the art and are well-described in the literature.3. Fragment Crystallizable Region, Fc

[0222] An Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are included.C. Polypeptides with antibody CDRs & Scaffolding Domains that Display the CDRs

[0223] Antigen-binding peptide scaffolds, such as complementarity-determining regions (CDRs), are used to generate protein-binding molecules in accordance with the aspects. Generally, a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and / or be easy to produce, express, and purify. Skerra, J Mol Recognit, 13: 167-87 (2000).

[0224] The protein scaffolds can be sourced from, but not limited to: fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratricopeptide repeat”. Such proteins are described in US Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. W02006 / 056464, each of which are specifically incorporated herein by reference in their entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal NO synthase (PIN) may also be used.D. Antibody Binding

[0225] The term “selective binding agent” refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab', F(ab')2, single chain antibodies, peptides, peptide fragments and proteins.

[0226] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. “Immunologically reactive” means that the selective binding agent or antibody of interest will bind with antigens present in a biological sample. The term “immune complex” refers the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.1. Affinity / Avidity

[0227] The term “affinity” refers the strength with which an antibody or selective binding agent binds an epitope. In antibody binding reactions, this is expressed as the affinity constant (Ka or ka sometimes referred to as the association constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of the antibody toits antigen relative to the binding strength of the antibody to an unrelated amino acid sequence. Affinity can be expressed as, for example, 20- fold greater binding ability of the antibody to its antigen then to an unrelated amino acid sequence. As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and / or selective binding agent.

[0228] There are several experimental methods that can be used by one skilled in the art to evaluate the binding affinity of any given antibody or selective binding agent for its antigen. This is generally done by measuring the equilibrium dissociation constant (KD or Kd), using the equation KD = koff / kon = [A][B] / [AB], The term koff is the rate of dissociation between the antibody and antigen per unit time, and is related to the concentration of antibody and antigen present in solution in the unbound form at equilibrium. The term kon is the rate of antibody and antigen association per unit time, and is related to the concentration of the bound antigen-antibody complex at equilibrium. The units used for measuring the KD are mol / L (molarity, or M), or concentration. The Ka of an antibody is the opposite of the KD, and is determined by the equation Ka = 1 / KD. Examples of some experimental methods that can be used to determine the KD value are: enzyme-linked immunosorbent assays (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE). The affinity constant (Ka) of an antibody is the opposite of the KD, and is determined by the equation Ka = 1 / KD.

[0229] Antibodies deemed useful in certain aspects may have an affinity constant (Ka) of about, at least about, or at most about 106, 107, 108, 109, or 1010M or any range derivable therein. Similarly, in some aspects, antibodies may have a dissociation constant of about, at least about or at most about 10'6, 10'7, 10'8, 10'9, 10'10M, or any range derivable therein. These values are reported for antibodies discussed herein and the same assay may be used to evaluate the binding properties of such antibodies. An antibody of the disclosure is said to “specifically bind” its target antigen when the dissociation constant (KD) is = 108M. The antibody specifically binds antigen with “high affinity” when the KD is ^5* 109M, and with “very high affinity” when the KD is ^5* 1010M.2. Epitope Specificity

[0230] The epitope of an antigen is the specific region of the antigen for which an antibody has binding affinity. In the case of protein or polypeptide antigens, the epitope is the specificresidues (or specified amino acids or protein segment) that the antibody binds with high affinity. An antibody does not necessarily contact every residue within the protein. Nor does every single amino acid substitution or deletion within a protein necessarily affect binding affinity. For purposes of this specification and the accompanying claims, the terms “epitope” and “antigenic determinant” are used interchangeably to refer to the site on an antigen to which B and / or T cells respond or recognize. Polypeptide epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide. An epitope typically includes at least 3, and typically 5-10 amino acids in a unique spatial conformation.

[0231] Epitope specificity of an antibody can be determined in a variety of ways. One approach, for example, involves testing a collection of overlapping peptides of 15 amino acids spanning the full sequence of the protein and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids). The peptides are immobilized in separate wells of a microtiter dish. Immobilization can be accomplished, for example, by biotinylating one terminus of the peptides. This process may affect the antibody affinity for the epitope, therefore different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for the purposes of comparison. This is useful for identifying end-specific antibodies. Optionally, additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments. An antibody or antigen-binding fragment is screened for binding to each of the various peptides. The epitope is defined as a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding.3. Modification of Antibody Antigen-Binding Domains

[0232] It is understood that the antibodies of the present disclosure may be modified, such that they are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the epitopes of the present disclosure. Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.

[0233] As discussed herein, minor variations in the amino acid sequences of antibodies or antigen-binding regions thereof are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% sequence identity. In particular, conservative amino acid replacements are contemplated.

[0234] Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families based on the chemical nature of the side chain; e.g., acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). For example, it is reasonable to expect that an isolated replacement of a leucine moiety with an isoleucine or valine moiety, or a similar replacement of an amino acid with a structurally related amino acid in the same family, will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Standard ELISA, Surface Plasmon Resonance (SPR), or other antibody binding assays can be performed by one skilled in the art to make a quantitative comparison of antigen binging affinity between the unmodified antibody and any polypeptide derivatives with conservative substitutions generated through any of several methods available to one skilled in the art.

[0235] Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Standard methods to identify protein sequences that fold into a known three-dimensional structure are available to those skilled in the art; Dill and McCallum., Science 338:1042-1046 (2012). Several algorithms for predicting protein structures and the gene sequences that encode these have been developed, and many of these algorithms can be found at the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / guide / proteins / ) and at the Bioinformatics Resource Portal (onthe World Wide Web at expasy.org / proteomics). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure.

[0236] Framework modifications can be made to antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to a corresponding germline sequence.

[0237] It is also contemplated that the antigen-binding domain may be multi-specific or multivalent by multimerizing the antigen-binding domain with VH and VL region pairs that bind either the same antigen (multi-valent) or a different antigen (multi-specific).E. Chemical Modification of Antibodies

[0238] In some aspects, also contemplated are glycosylation variants of antibodies, wherein the number and / or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861). In certain aspects, antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N- linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. In other aspects, one or more new N-linked glycosylation sites are created. Antibodies typically have an N-linked glycosylation site in the Fc region.

[0239] Additional antibody variants include cysteine variants, wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0240] In some aspects, the polypeptides can be pegylated to increase biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the polypeptide. Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the disclosure to obtain PEGylated derivatives of antibodies. See, e.g., EP 0 154 316 and EP 0 401 384. In some aspects, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols, and polyvinyl alcohols. As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins.1. Conjugation

[0241] Derivatives of the antibodies and antigen binding fragments that are described herein are also provided. The derivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment. The derivatized antibody can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses).

[0242] Optionally, an antibody or an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins. In some aspects, polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule. See, e.g., EP 0322094 and EP 0 486 525. In some aspects, the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen. In some aspects, the polypeptides may also be conjugated to a therapeutic agent to provide a therapy incombination with the therapeutic effect of the polypeptide. Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as a siRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.

[0243] In some aspects, disclosed are antibodies and antibody-like molecules that are linked to at least one agent to form an antibody conjugate or payload. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like. By contrast, a reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands. a. Conjugate Types

[0244] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. “Detectable labels” are compounds and / or elements that can be detected due to their specific functional properties, and / or chemical characteristics, the use of which allows the antibody to be detected, and / or further quantified if desired. Examples of detectable labels include, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. Particular examples of labels are, but not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and a- or P-galactosidase. Antibody conjugates include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme to generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The uses of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference. Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).

[0245] In some aspects, contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In this way, the agent of interest can be targeted directly to cells bearing cell surface antigen. The antibody and agent may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. Various linkers, known in the art, can be employed in order to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein. In one aspect, an antibody may be conjugated to various therapeutic substances in order to target the cell surface antigen. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens.

[0246] In antibody drug conjugates (ADC), an antibody (Ab) is conjugated to one or more drug moieties (D) through a linker (L). The ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Antibody drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of theconjugate. In yet another aspect, the antibody may be conjugated to a “receptor” (such as streptavidin) for utilization in tumor or cancer cell pre-targeting wherein the antibody -receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0247] Examples of an antibody-drug conjugates known to a person skilled in the art are pro-drugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605- 614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26: 151-172 (1997); U.S. Pat. No. 4,975,278). In contrast, systematic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the target tumor cells (Baldwin et al., Lancet 1 :603-5 (1986); Thorpe, (1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” In: Monoclonal Antibodies ‘84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506). Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21 : 183-87 (1986)).

[0248] In certain aspects, ADC include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Antibody-containing fusion proteins may comprise peptides added to facilitate purification or identification of the antibody (e.g., poly- His). An antibody polypeptide also can be linked to the FLAG® (Sigma-Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio / Technology 6: 1204 (1988), and U.S. Pat. No. 5,011,912. Oligomers that contain one or more antibody polypeptides may be employed as antagonists. Oligomers may be in the form of covalently linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibody polypeptides are contemplated for use. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. In certain aspects, oligomers comprise multiple antibody polypeptides joined via covalent or non-covalent interactions between peptide moi eties fused to the antibody polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptidesderived from antibodies are among the peptides that can promote oligomerization of antibody polypeptides attached thereto, as described in more detail below. b. Conjugation Methodology

[0249] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and / or tetrachloro-3 -6 -diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bos(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). In some aspects, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., 1987).II. Antibody Production

[0250] Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, for purification, and for use as therapeutics are well known in the art as disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745 (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). These antibodies may be polyclonal ormonoclonal antibody preparations, monospecific antisera, human antibodies, hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question. In certain aspects, polypeptides, peptides, and proteins and immunogenic fragments thereof for use in various aspects can also be synthesized in solution or on a solid support in accordance with conventional techniques. See, for example, Stewart and Young, (1984); Tarn et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.

[0251] Other methods of making antibodies are through molecular display technologies. These include technologies such as phage display and yeast surface display, have been the major driving force of the development (Winter, 1994; Boder, 2000; Viti, 2000; Bradbury, 2011). These enable display of single-chain Fv (scFv) and Fab fragments on the surface of microorganisms and links the phenotype of a variant to its cognate DNA and thus amino acid sequence (McCafferty, 1990; Kang, 1991; Barbas, 1991). Using these techniques, it is possible to produce a library of an antibody fragment that contains billions of variants and identify variants with a desired binding function from the library in a HTP manner.

[0252] In the standard antibody architecture, six surface loops, three each from the heavychain and light-chain variable domains constitute the complementarity determining regions (CDRs). Thus, the antibody and its fragments can be considered as "molecular scaffolds" for presenting CDRs. The sequence diversity of CDRs is the primary source of antibody affinity and specificity. In recombinant antibody libraries, the diversity of CDRs can be either derived from natural immune systems or generated by random mutagenesis, and Fv libraries of both types have been successfully used (Barbas, 1992; Knappik, 2000).

[0253] Yeast surface display is another important technical development in antibody engineering. Although the library size achievable with this technique is much smaller than that with phage display, yeast surface display coupled with fluorescence activated cell sorter (FACS) allows for HTP, quantitative characterization of antibody affinity, specificity and expression efficiency (Boder, 2000). The major strengths of phage display include the large size of a library and rapid completion of the selection and propagation cycle (< 2 days). Yeast display provides a highly quantitative readout of binding and expression levels using FACS and the ability to precisely define the selection stringency based on equilibrium and kinetic binding parameters as well as expression levels. The integration of these two display methods can provide for an optimal selection of clones that not only are high affinity binders but are stable and express well.

[0254] The end products of recombinant antibody screening are the genes encoding Fv and Fab fragments with desired binding characteristics. Bacterial expression systems for Fv and Fab with good yields have been established [Borrebaeck, 1992], Thus, the phage display clones can be quickly reformatted into an expression cassette for the production of soluble Fv and Fab fragments.

[0255] Library design: At least 15- 20 residues of CDR residues contribute to antibody binding to its target. Full combinatorial randomization of this many sites together far exceeds the limits of phage and yeast display library diversity. Since the number of sites cannot be significantly reduced, new library strategies have been developed that reduce the 20 natural amino acid genetic code to a smaller subset, while maintaining high functional diversity (Fellouse, 2007). These libraries using a greatly reduced genetic code can produce high affinity Fab and scFv molecules. Although several reduced genetic code libraries have been constructed and tested, the concept has not been optimized for different target classes and especially for the smaller scaffold camelids VHH and FN3 domains (D2). For screening of these novel libraries, we will combine phage display and yeast surface display for optimal efficiency. Humanized antibody scaffolds- In molecular display approaches only the variable regions of the antigen binding loops (CDRs) of the Fab domain are generally diversified. The main architecture of the domain is held constant. This main architecture can be based on a fully human antibody scaffold. This circumvents the need to humanize the scaffold, as is required by methods involving animal immunizations.

[0256] Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation, or overall functional properties relative to being a treatment for infection. Thus, monoclonal antibodies may have alterations in the amino acid sequence of CDRs, including insertions, deletions, or substitutions with a conserved or non-conserved amino acid.

[0257] The immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants that may be used in accordance with aspects include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur- MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Exemplary adjuvants may include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants,and / or aluminum hydroxide adjuvant. In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), such as but not limited to, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines such as P-interferon, IL-2, or IL- 12, or genes encoding proteins involved in immune helper functions, such as B-7.A phage-display system can be used to expand antibody molecule populations in vitro. Saiki, et al., Nature 324: 163 (1986); Scharf et al., Science 233: 1076 (1986); U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol. 254:392 (1995); Barbas, III et al., Methods: Comp. Meth Enzymol. (1995) 8:94; Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991).III. Antibodies, Antigen Binding Fragments, and Polypeptides

[0258] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. The term polypeptide also includes an antibody fragment described herein as well as antibody domains, such as HCDR1 (Heavy chain complementarity determing region 1), HCDR2 (Heavy chain complementarity determing region 2), HCDR3 (Heavy chain complementarity determing region 3), LCDR1 (Light chain complementarity determing region 1), LCDR2 (Light chain complementarity determing region 2), LCDR3 (Light chain complementarity determing region 3), HFRW1 (Heavy chain framework region 1), HFRW2 (Heavy chain framework region 2), HFRW3 (Heavy chain framework region 3), HFRW4 (Heavy chain framework region 4), LFRW1 (Light chain framework region 1), LFRW2 (Light chain framework region 21), LFRW3 (Light chain framework region 3), LFRW4 (Light chainframework region 4), VH (Heavy chain variable region), VL (Light chain variable region), CH (Heavy chain constant region), or CL (Light chain constant region).

[0259] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0260] In certain aspects the size of an antibody, antigen binding fragment, protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 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, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0261] The antibody, antigen binding fragment, polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 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, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 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, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein, of a polypeptide of Table 1 or Table 2.

[0262] In some aspects, the antibody, antigen binding fragment, protein, or polypeptide may comprise amino acids 1 to 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, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343,, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419,, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571,, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609,, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647,, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685,, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704,, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723,, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742,, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761,, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780,, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799,, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837,, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875,, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894,, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913,, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932,, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951,, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970,971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000, (or any derivable range therein) of a polypeptide of Table 1 or Table 2.

[0263] In some aspects, the antibody, antigen binding fragment, or polypeptide may comprise 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, 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, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613,614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632,633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651,652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670,671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689,690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708,709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727,728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746,747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765,766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784,785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803,804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841,842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879,880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898,899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917,918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936,937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955,956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974,975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993,994, 995, 996, 997, 998, 999, or 1000, (or any derivable range therein) contiguous amino acids of a polypeptide of Table 1 or Table 2.

[0264] In some aspects, the antibody, antigen binding fragment, protein, or polypeptide may comprise at least, at most, or exactly 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, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455,, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550,, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588,, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607,, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645,, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721,, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759,, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778,, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797,, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816,, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854,855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873,874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911,912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949,950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) contiguous amino acids of a polypeptide of Table 1 or Table 2 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of a polypeptide of Table 1 or Table 2.

[0265] In some aspects there is a nucleic acid molecule, antibody, antigen binding fragment, protein, or polypeptide starting at position 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, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547,548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642,643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699,700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718,719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737,738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756,757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775,776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813,814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851,852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889,890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908,909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927,928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965,966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984,985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 of any polypeptide of Table 1 or Table 2 and comprising at least, at most, or exactly 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,, 35, 36, 37, 38, 39, 40, 41. 42, 43, 44, 45 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,, 60, 61, 62, 63, 64, 65, 66 67, 68, 69, 10 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,6, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,5, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,4, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,3, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,2, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,1, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,0, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,9, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,8, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,7, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,6, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,5, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,4, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,3, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,2, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,1, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,0, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,9, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,8, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,7, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,6, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,5, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543,4, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,3, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581,2, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600,1, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,0, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,9, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657,8, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676,7, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714,715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733,734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752,753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771,772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809,810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847,848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885,886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904,905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923,924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942,943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961,962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980,981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) contiguous amino acids or nucleotides of any polypeptide of Table 1 or Table 2.

[0266] In some aspects, the amino acid at position 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, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, or 400 of the heavy chain, light chain, VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFRW1, HFRW2, HFRW3, HFRW4, LFRW1, LFRW2, LFRW3, or LFRW4 identified in Table 1 or Table 2 is substituted with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0267] In some aspects, a polypeptide (e.g., antibody, antibody fragment, Fab, etc.) of the disclosure comprises a CDR that is at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) in sequence to one polypeptide of Table 1 or Table 2. In some aspects, a polypeptide comprises 1, 2, and / or 3 domains from one of SEQ ID NOs: 1-103. The CDR may be one that has been determined by Kabat, IMGT, or Chothia. In further aspects, a polypeptide may have CDRs that have 1, 2, and / or 3 amino acid changes (e.g., addition of 1 or 2 amino acids, deletions of 1 or 2 amino acids, substitution) with respect to these 1, 2, or 3 CDRs. In some aspects, a polypeptide comprises additionally or alternatively, an amino acid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to the amino acid sequence of the variable region that is not a CDR sequence, i.e., the variable region framework.

[0268] From amino to carboxy terminus the CDRs are CDR1, CDR2, and CDR3. In some aspects, a polypeptide may have CDRs that have 1, 2, and / or 3 amino acid changes (e.g., addition of 1 or 2 amino acids, deletions of 1 or 2 amino acids, substitution) with respect to CDR1, CDR2, or CDR3. In some aspects, the CDRs of Table 1 or Table 2 may further comprise 1, 2, 3, 4, 5, or 6 additional amino acids at the amino or carboxy terminus of the CDR, The additional amino acids may be from the heavy and / or light chain framework regions of Table 1 or Table 2, that are shown as immediately adjacent to the CDRs. Accordingly, aspects relate to polypeptides comprising an HCDR1 (i.e., CDR-H1), HCDR2 (i.e., CDR-H2), HCDR3( i.e., CDR-H3), LCDR1 (i.e., CDR-L1), LCDR2 (i.e., CDR-L2), and / or LCDR3 (i.e., CDR-L3) with at least or at most or exactly 1, 2, 3, 4, 5, 6 or 7 amino acids at the amino end of the CDR or at the carboxy end of the CDR, wherein the additional amino acids are the 1, 2, 3, 4, 5, 6, or 7 amino acids of Table 1 or Table 2 that are shown as immediately adjacent to the CDRs. Other aspects relate to antibodies comprising one or more CDRs, wherein the CDR is a fragment ofTable 1 or Table 2 and wherein the fragment lacks 1, 2, 3, 4, or 5 amino acids from the amino or carboxy end of the CDR. In some aspects, the CDR may lack one, 2, 3, 4, 5, 6, or 7 amino acids from the carboxy end and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the framework region of the amino end of the CDR. In some aspects, the CDR may lack one, 2, 3, 4, 5, 6, or 7 amino acids from the amino end and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from the framework region of the carboxy end of the CDR. In further aspects, an antibody may be alternatively or additionally humanized in regions outside the CDR(s) and / or variable region(s). In some aspects, a polypeptide comprises additionally or alternatively, an amino acid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to the amino acid sequence of the variable region that is not a CDR sequence, i.e., the variable region framework.

[0269] In other aspects, a polypeptide or protein comprises 1, 2, 3, 4, 5, or 6 CDRs from either or both of the light and heavy variable regions of Table 1, and 1, 2, 3, 4, 5, or 6 CDRs may have 1, 2, and / or 3 amino acid changes with respect to these CDRs. In some aspects, parts or all of the antibody sequence outside the variable region have been humanized. A protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and / or 1 or 2 polypeptides similar to a light chain polypeptide.

[0270] The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0271] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).Table 1: Antibody and Antigen Binding Domain SequencesTable 2: Bi-Specific and Antigen Binding Domain SequencesTable 3: Summary of Antibody ClonesTable 4: Summary of Bi-Specific Clones1. Variant Polypeptides

[0272] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0273] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0274] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 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, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0275] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0276] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0277] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0278] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0279] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.2. Considerations for Substitutions

[0280] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further aspects, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0281] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interactionof the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain aspects, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the disclosure, those that are within ±1 are included, and in other aspects of the disclosure, those within ±0.5 are included.

[0282] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (—0.5); histidine (—0.5); cysteine (—1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain aspects, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other aspects, those which are within ±1 are included, and in still other aspects, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0283] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0284] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of suchinformation, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein_structure.

[0285] In some aspects of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such aspects, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).IV. Nucleic Acids

[0286] In certain aspects, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding peptides and polypeptides of the disclosure, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. Nucleic acids encoding fusion proteins that include these peptides are also provided. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0287] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0288] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0289] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0290] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200,250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.A. Hybridization

[0291] The nucleic acids that hybridize to other nucleic acids under particular hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5* sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6* SSC, and a hybridization temperature of 55° C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42° C), and washing conditions of 60° C. in 0.5* SSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6* SSC at 45° C., followed by one or more washes in 0.1 * SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequence that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other typically remain hybridized to each other.

[0292] The parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are herein incorporated by reference in their entirety for all purposes) and can be readily determined bythose having ordinary skill in the art based on, for example, the length and / or base composition of the DNA.B. Mutation

[0293] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigenic peptide or polypeptide) that it encodes. Mutations can be introduced using any technique known in the art. In one aspect, one or more particular amino acid residues are changed using, for example, a site- directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0294] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, eg., Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.C. Probes

[0295] In another aspect, nucleic acid molecules are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences. A nucleic acid molecule can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of a given polypeptide.

[0296] In another aspect, the nucleic acid molecules may be used as probes or PCR primers for specific nucleic acid sequences. For instance, a nucleic acid molecule probe may be used in diagnostic methods or a nucleic acid molecule PCR primer may be used to amplify regions of DNA that could be used, inter alia, to isolate nucleic acid sequences for use in producing the engineered cells of the disclosure. In a preferred aspect, the nucleic acid molecules are oligonucleotides.

[0297] Probes based on the desired sequence of a nucleic acid can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of interest. The probe can comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide.V. Polypeptide Expression

[0298] In some aspects, there are nucleic acid molecule encoding polypeptides, antibodies, or antigen binding fragments of the disclosure. The nucleic acid molecules may be used to express large quantities of polypeptides. If the nucleic acid molecules are derived from a nonhuman, non-transgenic animal, the nucleic acid molecules may be used for humanization of the antibody or TCR genes.A. Vectors

[0299] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigenbinding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody heavy and / or light chain, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0300] To express the polypeptides or peptides of the disclosure, DNAs encoding the polypeptides or peptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In some aspects, a vector that encodes a functionally complete human TCR alpha or TCR beta sequence with appropriate restriction sites engineered so that any variable sequence or CDR1, CDR2, and / or CDR3 can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Suchsequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.B. Expression Systems

[0301] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with an aspect to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.C. Methods of Gene Transfer

[0302] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolauet al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.VI. Cancer Therapy

[0303] In some aspects, the disclosed methods comprise administering a cancer therapy to a subject or patient. The cancer therapy may comprise one or more of the antibodies or antigenbinding fragments disclosed herein. In some aspects, the cancer therapy comprises an antibody, antigen-binding fragment, and / or bi-specific antibody capable of binding a Vy9V52 T cell. The cancer therapy may be chosen based on an expression level measurement, alone or in combination with the clinical risk score calculated for the subject. The cancer therapy may be chosen based on a genotype of a subject. The cancer therapy may be chosen based on the presence or absence of one or more polymorphisms in a subject. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some aspects, the cancer therapy comprises an immunotherapy, which may be a checkpoint inhibitor therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

[0304] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is a Stage I cancer. In some aspects, the cancer is a Stage II cancer. In some aspects, the cancer is a Stage III cancer. In some aspects, the cancer is a Stage IV cancer.

[0305] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor,malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0306] In some aspects, the cancer is aggressive cancer. In some aspects, the cancer is Stage I cancer. In some aspects, the cancer is Stage II cancer (e.g., IIA, IIB, IIC). In some aspects, the cancer is Stage III cancer (e.g., IIIA, IIIB, IIIC). In some aspects, the cancer is Stage IV cancer (e.g., IVA, IVB).

[0307] Methods may involve the determination, administration, or selection of an appropriate cancer “management regimen” and predicting the outcome of the same. As used herein the phrase “management regimen” refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and / or duration of a treatment) provided to a subject in need thereof e.g., a subject diagnosed with cancer).A. Immunotherapy

[0308] In some aspects, the methods comprise administration of a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour- associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells bytargeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below.1. Inhibition of co-stimulatory molecules

[0309] In some aspects, the immunotherapy comprises an inhibitor of a co-stimulatory molecule. In some aspects, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.2. Dendritic cell therapy

[0310] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0311] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0312] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0313] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0314] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendriticcells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.3. CAR-T cell therapy

[0315] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0316] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signalling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0317] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some aspects, the CAR-T therapy targets CD19.4. Cytokine therapy

[0318] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0319] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IF NX).

[0320] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.5. Adoptive T-cell therapy

[0321] Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death.

[0322] Multiple ways of producing and obtaining tumour targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.

[0323] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, aspects of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some aspects, the patient is one that has been determined to be resistant to a therapy described herein. In some aspects, the patient is one that has been determined to be sensitive to a therapy described herein.B. Checkpoint Inhibitors and Combination Treatment

[0324] Aspects of the disclosure may include administration of immune checkpoint inhibitors, which are further described below.1. PD-1, PDL1, and PDL2 inhibitors

[0325] PD -1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumorcells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0326] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0327] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7- 1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0328] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS- 936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0329] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0330] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.2. CTLA-4, B7-1, and B7-2

[0331] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0332] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0333] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0334] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0335] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.C. Combination therapies

[0336] In some aspects, the methods may further comprise administration of an additional therapy such as radiotherapy, cancer immunotherapy, oncolytic virus, chemotherapies, hormone therapies, or surgery. Specific chemotherapies include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine,pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhy diazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane), antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”), pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode-oxyuridine; FudR).VII. Pharmaceutical compositions

[0337] The present disclosure includes methods for treating disease and modulating immune responses in a subject in need thereof. The disclosure includes cells that may be in the form of a pharmaceutical composition that can be used to induce or modify an immune response.

[0338] Administration of the compositions according to the current disclosure will typically be via any common route. This includes, but is not limited to parenteral, orthotopic, intradermal, subcutaneous, orally, transdermally, intramuscular, intraperitoneal, intraperitoneally, intraorbitally, by implantation, by inhalation, intraventricularly, intranasally or intravenous injection. In some aspects, compositions of the present disclosure (e.g., compositions comprising TCR-binding polypeptides) are administered to a subject intravenously.

[0339] Typically, compositions and therapies of the disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying. The quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0340] The manner of application may be varied widely. Any of the conventional methods for administration of pharmaceutical compositions comprising cellular components are applicable. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.

[0341] In many instances, it will be desirable to have multiple administrations of at most or at least 3, 4, 5, 6, 7, 8, 9, 10 or more. The administrations may range from 2-day to 12-week intervals, more usually from one to two week intervals.

[0342] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. The pharmaceutical compositions of the current disclosure are pharmaceutically acceptable compositions.

[0343] The compositions of the disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions and the preparations can also be emulsified.

[0344] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0345] Sterile injectable solutions are prepared by incorporating the active ingredients (e.g., polypeptides of the disclosure) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0346] An effective amount of a composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration,intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0347] The compositions and related methods of the present disclosure, particularly administration of a composition of the disclosure may also be used in combination with the administration of additional therapies such as the additional therapeutics described herein or in combination with other traditional therapeutics known in the art.

[0348] The therapeutic compositions and treatments disclosed herein may precede, be cocurrent with and / or follow another treatment or agent by intervals ranging from minutes to weeks. In aspects where agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more agents or treatments substantially simultaneously (i.e., within less than about a minute). In other aspects, one or more therapeutic agents or treatments may be administered or provided within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days,3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any range derivable therein, prior to and / or after administering another therapeutic agent or treatment.

[0349] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0350] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0351] In some aspects, the therapeutically effective or sufficient amount of the immune checkpoint inhibitor, such as an antibody and / or microbial modulator, that is administered to a human will be in the range of about 0.01 to about 50 mg / kg of patient body weight whether by one or more administrations. In some aspects, the therapy used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily, for example. In one aspect, a therapy described herein is administered to a subject at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21 -day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of this therapy is easily monitored by conventional techniques.

[0352] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another aspect, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 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, 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, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 M or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0353] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0354] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.VIII. Detectable Labels

[0355] In some aspects of this disclosure, it will be useful to detectably or therapeutically label a Fab polypeptide or protein G Fab-binding domain. Methods for conjugating polypeptides to these agents are known in the art. For the purpose of illustration only, polypeptides can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled polypeptides can be used for diagnostic techniques, either in vivo, or in an isolated test sample or in methods described herein.

[0356] As used herein, the term "label" intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal uponexpression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0357] Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

[0358] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).

[0359] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0360] Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens / polypeptides, e.g., rhodamine / anti-rhodamine, biotin / avidin and biotin / strepavidin.

[0361] The coupling of polypeptides to low molecular weight haptens can increase the sensitivity of the antibody in an assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten polypeptides. See, Harlow and Lane (1988) supra.IX. Sample Preparation

[0362] In certain aspects, methods involve obtaining or evaluating a sample from a subject. The sample may include a sample obtained from any source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0363] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[0364] The sample may be obtained by methods known in the art. In certain aspects the samples are obtained by biopsy. In other aspects the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example esophagus) and one or more samples from another specimen (for example serum) may be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type (e.g. esophagus) and one or more samples from- I l l -another specimen (e.g. serum) may be obtained at the same or different times. Samples may be obtained at different times are stored and / or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[0365] In some aspects the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0366] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some aspects, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0367] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In one aspect, the sample is a fine needle aspirate of a tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[0368] In some aspects of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0369] In some aspects of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain ameans for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and / or adequacy are provided.

[0370] In some aspects, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample.X. Host Cells

[0371] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include both freshly isolated cells and ex vivo cultured, activated or expanded cells. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

[0372] In certain aspects transfection can be carried out on any prokaryotic or eukaryotic cell. In some aspects electroporation involves transfection of a human cell. In other aspects electroporation involves transfection of an animal cell. In certain aspects transfection involves transfection of a cell line or a hybrid cell type. In some aspects the cell or cells being transfected are cancer cells, tumor cells or immortalized cells. In some instances tumor, cancer, immortalized cells or cell lines are induced and in other instances tumor, cancer, immortalized cells or cell lines enter their respective state or condition naturally. In certain aspects the cellsor cell lines can be A549, B-cells, B16, BHK-21, C2C12, C6, CaCo-2, CAP / , CAP-T, CHO, CHO2, CHO-DG44, CH0-K1, COS-1, Cos-7, CV-1, Dendritic cells, DLD-1, Embryonic Stem (ES) Cell or derivative, H1299, HEK, 293, 293T, 293FT, Hep G2, Hematopoietic Stem Cells, HOS, Huh-7, Induced Pluripotent Stem (iPS) Cell or derivative, Jurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, Mesenchymal Cells, Min-6, Monocytic cell, Neuro2a, NIH 3T3, NIH3T3L1, K562, NK-cells, NSO, Panc-1, PC12, PC-3, Peripheral blood cells, Plasma cells, Primary Fibroblasts, RBL, Renca, RLE, SF21, SF9, SH-SY5Y, SK-MES- 1, SK-N-SH, SL3, SW403, Stimulus-triggered Acquisition of Pluripotency (STAP) cell or derivate SW403, T-cells, THP-1, Tumor cells, U2OS, U937, peripheral blood lymphocytes, expanded T cells, hematopoietic stem cells, or Vero cells.XI. Kits

[0373] Certain aspects of the present disclosure also concern kits containing compositions of the disclosure or compositions to implement methods of the disclosure. In some aspects, kits can be used to detect the presence of a cancer cell in a sample. In certain aspects, a kit contains, contains at least or contains at most 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, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein. In some aspects, a kit contains one or more polypeptides capable of binding to a TCR (including a Vy9V52 TCR), including polypeptides disclosed herein. For example, a kit may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antibodies or antibody fragments (e.g., Fabs) disclosed herein for detecting a TCR (including a Vy9V52 TCR), in some cases for detecting TCR (including a Vy9V52 TCR) on expressed by a T cell. In some aspects, a kit comprises a detection pair. In some aspects, a kit comprises an enzyme. In some aspects, a kit comprises a substrate for an enzyme.

[0374] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0375] Individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

[0376] Kits for using probes, synthetic nucleic acids, nonsynthetic nucleic acids, and / or inhibitors of the disclosure for prognostic or diagnostic applications are included as part of the disclosure. In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit aspects.

[0377] Kits may further comprise instructions for use. For example, in some aspects, a kit comprises instructions for detecting a TCR (including a Vy9V52 TCR) in a sample.

[0378] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different aspects may be combined. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.XII. Examples

[0379] The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Generation and Testing of Vy9V62 T-cell Receptor Binding Antibodies And Antigen Binding Fragments.

[0380] Aspects herein relate to the design and development of pan-specific antibodies capable of recognizing the Vy9V52 T-cell receptor (TCR) and inducing T-cell activation through allosteric triggering. Employing a competitive phage display approach, aspects herein successfully generat a panel of Vy9V52-specific antibodies that efficiently triggered T-cell activation, ultimately leading to the elimination of cancer cells. The high-resolution crystal structure of the Vy9V52 TCR in complex with the T7 Fab, resolved at 2.16 , provides valuable molecular insights into the pan-specific recognition properties of the T7 Fab. Notably, this structure reveals the binding of T7 Fab to a conserved motif present in Vy9V52 TCRs derived from diverse patient populations, underscoring its potential utility in the development of novel immunotherapeutic strategies targeting Vy9V52 T cells.

[0381] To conclude, the inventors have generated a panel of <z-Vy9V52 antibodies. The T7 Fab has a very high affinity and acceptable specificity (with a preference to 52 chain). The T7Fab has an outstanding developability profile due to a very short and not hydrophobic CDR- H3. The inventors solved the crystal structure of T7 Fab with Vy9V52 TCR (at 2.16A). The T7 Fab binds to a public domain of CDR3 on Vy9V52 T cells (and does not disturb the interaction with BTN2A1). The T7 Fab plug-and-play BiTE was efficient to induce cancer cells killing by Vy9V52 T cells.Example 2 - Development of Synthetic Human Monoclonal Antibodies that bind to Vy9V62 T cells.

[0382] The sequences of Vy9 and V52-chain with C-terminal Avi-tag were cloned separately into the pSCSTa expressing vector. The Vy9V52 TCR, engineered with a C-terminal Avi-tag, was expressed by co-transfection of both chains in mammalian Expi293 cells using the ExpiFectamine 293 Transfection Kit, following the manufacturer’s protocol. Subsequent biotinylation of the protein was performed via the Avi-tag using the BirA enzyme (Avidity, Cat. number: BirA500). Antibodies were generated through phage display. Specifically, five rounds of selection were conducted using a highly diverse (IO10) synthetic library (Miller et al., 2012), following established protocols (Fellouse et al., 2007; Paduch et al., 2013) (Figure 1 A). To enrich for high-affinity binders, the antigen concentration was systematically decreased from 1 pM in the first round to 1 nM in the fifth round. Phage ELISA screening of 480 clones identified seven unique high-affinity variants, designated T1-T7. Protein ELISA analysis revealed three distinct populations of binders: Tl, T5, and T6, which preferentially bind the V52 chain; T4, which binds the Vy9 chain; and T2, T3, and T7, which selectively bind the Vy9V52 TCR (Figure IB). The Fabs were further characterized, and their binding affinities were determined by Surface Plasmon Resonance (SPR) (Figure 1G). All antibodies exhibited low nanomolar binding constants (KD) and slow dissociation rates.Example 3 - Fab production.

[0383] Fabs were expressed in the periplasm of E. coli BL21 cells for 4 hours at 37°C post-induction with 1 mM IPTG at ODeoo= 0.8-1. The cells were harvested by sonication. After centrifugation, the supernatant was applied to the protein GF affinity column (Slezak et al. 2025). Proteins were eluted from the column with 0.1 M glycine, pH 2.6, and neutralized with IM Tris-HCl, pH 8.5, and dialyzed overnight into PBS.Example 4 - IgG production.

[0384] Full-length IgG were expressed in Expi293 cells at 2xl06cells / mL. The heavy (IgGl scaffold) and light chain (Kappa) were co-transfected using ExpiFectamine 293transfection reagent (Gibco, Cat. number: A14525) according to the manufacture’s recommendation. Five days post-transfection the supernatant was collected by centrifugation and IgG was purified using a protein GF affinity column (Slezak et al. 2025). Proteins were eluted from the column with 0.1 M glycine, pH 2.6, and neutralized with IM Tris-HCl, pH 8.5, and dialyzed overnight into PBS.Example 5 - T7 BiTE production.

[0385] As a proof of concept, BiTE targeting HER2 and Vy9V52 TCR was designed by cloning Her2 scFv with T7 scFv into the pSCSTa vector. The BiTE was expressed in Expi293 cells at 2xl06cells / mL using ExpiFectamine 293 transfection reagent (Gibco, Cat. number: A14525) according to the manufacture’s recommendation. Five days post-transfection the supernatant was collected by centrifugation and IgG was purified using a TALON resin (TAKARA, Cat. No. 635501). Proteins were eluted from the column with 150 mM imidazole in buffer A (20 mM TRIS pH 8.0, 150 mM NaCl, 10% glycerol) and dialyzed overnight into PBS.Example 6 - T7 Bi-specific IgG (bi-IgG) production.

[0386] Bi-specific IgG molecules were engineered by combining Her2-specific IgG with a T7 single-chain variable fragment (scFv) fused to the Fc region, incorporating knobs- into-holes mutations as described by Von Kreudenstein et al. (2013). Expression of the bi- specific IgG was performed in Expi293 cells at a density of 2x 1062x 106 cells / mL. Constructs included a heavy chain bearing the ZWA mutation (IgGl scaffold), a kappa light chain, and an Fc-scFv fusion with ZWB mutations. These components were co-transfected using the ExpiFectamine 293 transfection reagent (Gibco, Cat. No. A14525) following the manufacturer’s protocol. Five days post-transfection, supernatants were harvested by centrifugation. The bi-specific IgG was subsequently purified via a protein GF affinity column as described by Slezak et al. (2025). Elution was achieved with 0.1 M glycine at pH 2.6, followed by immediate neutralization with 1 M Tris-HCl at pH 8.5. The purified proteins were then dialyzed overnight into PBS.Example 7 - a-Vy9V62 Tabs binding by ELISA assay.

[0387] The ELISA assay was performed to validate the <z-Vy9V52 Fabs binding to Vy9V52 TCR. 50 nM of biotinylated Vy9V52 TCR was immobilized on a 96-well neutravidin- coated plate (Geiner), followed by extensive blocking with BSA. Phage or purified Fabs were incubated on the plate for 15 minutes, washed three times, and incubated with either anti -Ml 3phage (Abeam) or anti-human Fab (Jackson Immunoresearch) antibody conjugated with HRP (in 1 :5000 dilution in PBST) for 20 minutes at room temperature. The plates were then washed, developed with TMB substrate (Thermo Scientific), and quenched with 10% H3PO4, followed by the absorbance at A450 determination.Example 8 - Confirmation of T7 Fab Binding to Vy9V62 TCR by Size Exclusion Chromatography.

[0388] Binding of T7 Fab to Vy9V52 TCR was validated using size exclusion chromatography. Purified Vy9V52 TCR was incubated with a 1.2-fold molar excess of T7 Fab on ice for 30 minutes. Samples of Vy9V52 TCR alone and the Vy9V52 TCR-T7 Fab complex were subsequently analyzed using a Superdex 200 column. Complex formation was confirmed by a shift in retention volume, indicative of successful binding of T7 Fab to Vy9V52 TCR (Figure ID).Example 9 - Thermal Stability Enhancement of Vy9V62 TCR Upon T7 Fab Binding Assessed by Differential Scanning Fluorimetry.

[0389] Thermal stabilities of Vy9V52 TCR and its complex with T7 Fab were assessed using differential scanning fluorimetry (DSF). Samples were prepared in triplicate at a protein concentration of 4 pM, utilizing Sypro Orange dye (Invitrogen, Cat. No. S6650). Thermal melts were conducted by heating the samples from 25 °C to 95 °C, with temperature increments of 0.5 °C every 30 seconds. Formation of the Vy9V52 TCR-T7 Fab complex resulted in a 1 °C increase in the thermal stability of the TCR, indicating enhanced structural stability upon Fab binding (Figure IE).Example 10 - a-Vy9V62 Fabs binding by Surface plasmon resonance analysis.

[0390] All Surface plasmon resonance (SPR) analyses were performed on a MASS-1 (Bruker) to determine the association and dissociation constants of <z-Vy9V52 Fabs. Target Vy9V52 TCR was immobilized via a lOx His-tag to a Ni-NTA sensor chip. Fabs in twofold dilutions were run as analytes at a 30 pl / min flow rate at 20°C. Sensograms were corrected through double referencing, and a 1 : 1 binding model fit was done using Sierra Analyzer (Bruker).Example 11 - a-Vy9V62 Fabs binding to Jurkat cells expressing Vy9V62 TCR.

[0391] Flow cytometry analysis was performed to determine the Fabs binding to the Jurkat cell lines expressing Vy9V52 TCR (Jrt-3.3 cells). <z-Vy9V52 Fabs were incubated with cellsfor 30 min at 4 °C and detected by an anti-human Fab secondary antibody conjugated to Alexa 647 (Jackson ImmunoResearch, Cat. No. 109-605-006) using a Flow cytometer (CytoFlex instrument; Beckman Coulter). Significant binding was observed compared to an isotype antibody recognizing SARS-CoV-2 RBD protein (Slezak and Kossiakoff, 2021) (Figure 3A).Example 12 - T7 Fab and IgG binding to PBMCs.

[0392] Flow cytometry analysis was performed to determine the T7 Fab and IgG binding to the PBMCs. T7 Fab or IgG were incubated with cells for 30 min at 4 °C and detected by an anti-human Fab secondary antibody conjugated to Alexa 647 (Jackson ImmunoResearch, Cat. No. 109-605-006) using a Flow cytometer (CytoFlex instrument; Beckman Coulter). Significant binding was observed compared to an isotope antibody recognizing SARS-CoV-2 RBD protein (Slezak and Kossiakoff, 2021) (Figure 3B, 3C).Example 13 - a-Vy9V62 Fabs T cell activation.

[0393] Flow cytometry analysis was performed to determine the <z-Vy9V52 Fabs T cell activation in PBMCs. The Fabs were incubated with cells for 8h at 37 °C, followed by the CD69 detection using anti-CD69 antibodies conjugated with APC (Biolegends, Cat. No. 310910) using a Flow cytometer (CytoFlex instrument; Beckman Coulter). Significant T cell activation was observed for T7 Fab (Figure 3D).Example 14 -T7 IgG binds to Vy9V62 T cell population in PBMCs.

[0394] Flow cytometry was employed to assess the binding of T7 IgG to the Vy9V52 T cell subset within peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with T7 IgG for 30 minutes at 4 °C. The Vy9V52 T cell population was identified using PE- conjugated anti-human TCR Vy9 antibody (BioLegend, Cat. No. 331307) and FITC- conjugated anti-human TCR V52 antibody (BioLegend, Cat. No. 331405). T7 IgG binding was detected with an Alexa 647-conjugated anti-human Fab secondary antibody (Jackson ImmunoResearch, Cat. No. 109-605-006) in both Vy9V52-positive and Vy9V52-negative cell populations using a CytoFlex flow cytometer (Beckman Coulter). Robust binding of T7 IgG was observed specifically in the Vy9V52-positive T cell population, whereas no detectable binding was observed in the Vy9V52-negative cells (Figure 3G).Example 15 - Vy9V62 T cell-mediated cytotoxicity against HCC-1954 cells via T7 plug- and-play BiTEs.

[0395] The initial cytotoxic efficacy of Vy9V52 T cells against HCC-1954 cancer cells was evaluated using a T7 Fab-based plug-and-play BiTE design (Figure 3A; Slezak et al., 2020). One day prior to the experiment, HCC-1954 cells were seeded into a 96-well plate. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cryopreserved in liquid nitrogen. On the day of the assay, PBMCs were thawed, and the y5 T cell population was increased via a / p TCR negative selection (StemCell, Cat. No. 17846). Pre-mixed plug-and- play BiTEs were added to the HCC-1954 and PBMC co-culture at a 1 :10 ratio, and the plate was incubated for 48 hours at 37°C. Following incubation, supernatants were collected and analyzed for lactate dehydrogenase (LDH) release (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity of the T7 plug- and-play BiTE in stimulating Vy9V52 T cells, yielding cytotoxicity levels comparable to those achieved by CD3-mediated Vy9V52 T cell engagement (Figure 4C).Example 16 - Her2-T7 BiTE-induced cytotoxicity of PBMCs against Cancer Cells.

[0396] The cytotoxic efficacy of the Her2-T7 BiTE in combination with full PBMCs against PC3 cells was assessed using an LDH release assay (Figure 5A). One day before the experiment, PC3 cells were seeded into a 96-well plate. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cryopreserved in liquid nitrogen. On the day of the assay, varying concentrations of Her2-T7 BiTE were added to the PC3 and PBMC cocultures at a 10: 1 effector-to-target ratio, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and analyzed for lactate dehydrogenase (LDH) release (Promega, Cat. No. G1781) according to the manufacturer’s protocol. As a positive control, a Her2 BiTE targeting CD3 was used, while the RBD1-T7 BiTE, specific for the SARS-CoV-2 RBD protein, served as a negative control. The results revealed potent cytotoxic activity of the Her2-T7 BiTE (Figure 5A).Example 17 - Her2-T7 BiTE-induced cytotoxicity of enriched Vy9V62 T cells against PC3 cells.

[0397] The cytotoxic efficacy of the Her2-T7 BiTE in combination with Vy9V52 T cells against PC3 cells was evaluated using a lactate dehydrogenase (LDH) release assay. Vy9V52 T cells were enriched from peripheral blood mononuclear cells (PBMCs) utilizing a commercial isolation kit (StemCell, Cat. No. 19255). One day prior to the assay, PC3 cells were seeded into 96-well plates. On the day of the experiment, varying concentrations of Her2- T7 BiTE were added to co-cultures of PC3 and Vy9V52 T cells at a target-to-effector (T:E)ratio of 1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and LDH release was quantified (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity mediated by the Her2-T7 BiTE (Figure 5B). As a positive control, a Her2 BiTE targeting CD3 was employed, while the RBD1-T7 BiTE, specific for the SARS-CoV-2 RBD protein, served as a negative control.Example 18 - Absence of IL-17 release following Her2-T7 BiTE-mediated co-culture of PBMCs and PC3 cells.

[0398] The secretion of interleukin- 17 (IL-17) by Vy9V52 T cells in response to Her2-T7 BiTE was evaluated in a 48-hour co-culture with PC3 cells. One day before the experiment, PC3 cells were seeded into a 96-well plate. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cryopreserved in liquid nitrogen. On the day of the assay, various concentrations of Her2-T7 BiTE were added to the PC3 and PBMC co-cultures at a 1 : 10 target-to-effector ratio, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and IL-17 levels were quantified using a commercial detection kit (Revvity, Cat. No. 62HIL17PET) according to the manufacturer’s instructions. As a positive control, a Her2-Okt3 BiTE targeting CD3 was included. The results demonstrated a lack of IL- 17 release when Vy9V52 T cell engagement was mediated by Her2-T7 BiTE (Figure 6A).Example 19 - Lack of IL-17 release by Vy9V62 T cells following Her2-T7 BiTE engagement in PC3 co-culture.

[0399] The secretion of interleukin- 17 (IL-17) by Vy9V52 T cells in response to Her2-T7 BiTE was assessed in a 48-hour co-culture with PC3 cells. One day before the experiment, PC3 cells were seeded into a 96-well plate. Vy9V52 T cells were enriched from peripheral blood mononuclear cells (PBMCs) using a commercial isolation kit (StemCell, Cat. No. 19255). On the day of the assay, various concentrations of Her2-T7 BiTE were added to PC3 and Vy9V52 T cell co-cultures at a 1 :3 target-to-effector ratio, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and IL-17 levels were quantified using a commercial detection kit (Revvity, Cat. No. 62HIL17PET) according to the manufacturer’s instructions. As a positive control, a Her2-Okt3 BiTE targeting CD3 was included. The results demonstrated an absence of IL- 17 release by Vy9V52 T cells under both activation conditions- via CD3 or Vy9V52 TCR engagement (Figure 6B).Example 20 - Her2-T7 BiTE induces robust IFN-y release by Vy9V62 T cells in PC3 coculture.

[0400] The release of interferon gamma (IFN-y) by Vy9V52 T cells in response to Her2- T7 BiTE was evaluated in a 48-hour co-culture with PC3 cells. One day before the experiment, PC3 cells were seeded into a 96-well plate. Vy9V52 T cells were enriched from peripheral blood mononuclear cells (PBMCs) using a commercial isolation kit (StemCell, Cat. No. 19255). On the day of the assay, varying concentrations of Her2-T7 BiTE were added to PC3 and Vy9V52 T cell co-cultures at a 1 :3 target-to-effector ratio, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and IFN-y levels were quantified using a commercial detection kit (Cisbio, Cat. No. 62HIFNGPEG), according to the manufacturer’s instructions. The results demonstrated robust IFN-y release triggered by Her2- T7 BiTE (Figure 6C). As a positive control, a Her2-Okt3 BiTE targeting CD3 was used.Example 21 - Incucyte-based analysis of Her2-T7 BiTE cytotoxicity using enriched Vy9V62 T cells.

[0401] The cytotoxic efficacy of T7 BiTE was assessed using the Incucyte S3 Imaging System. One day before the experiment, PC3 cells were seeded into a 96-well plate. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and Vy9V52 T cells were enriched using a commercial isolation kit (StemCell, Cat. No. 19255). The following day, various concentrations of BiTEs and Vy9V52 T cells were added at an effector-to-target ratio of 3 : 1. Cancer cell death was monitored over 70 hours by evaluating changes in cell confluence and by quantifying dead cells using a commercially available viability dye (Sartorius, Cat. No. 4632) with the Incucyte S3 Imaging System. As a negative control, RBD1-T7 BiTE, which targets the SARS-CoV-2 RBD protein, was utilized. The results demonstrate that Her2-T7 BiTE induces robust, concentration-dependent cytotoxicity against PC3 cells (Figure 7A-7C).Example 22 - Cytotoxic evaluation of Her2-T7 BiTE and bi-IgG using expanded Vy9V62 T cells and PC3 cancer cells.

[0402] The cytotoxic activities of Her2-T7 BiTE and Her2-T7 bi-IgG were evaluated usingPC3 cells and Vy9V52 T cells expanded in vitro. Peripheral blood mononuclear cells (PBMCs) were cultured for seven days in the presence of IL-2 and 0.1 nM T4 IgG to selectively expand the Vy9V52 T cell population. One day before the experiment, PC3 cells were seeded onto a 96-well plate. On the day of the experiment, varying concentrations of Her2-T7 BiTE or Her2- T7 bi-IgG were added to co-cultures of PC3 and Vy9V52 T cells at a target-to-effector ratio of1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and lactate dehydrogenase (LDH) release was quantified using a commercial assay (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity mediated by both Her2-T7 BiTE and Her2-T7 bi-IgG (Figure 11 A). As a positive control, a Her2 BiTE targeting CD3 was included, while the isotype control (RBD1- T7 BiTE, specific for the SARS-CoV-2 RBD protein) served as a negative control.Example 23 - Cytotoxic evaluation of Her2-T7 BiTE and bi-IgG using expanded Vy9V62 T cells and OVCAR-3 cancer cells.

[0403] The cytotoxic activities of Her2-T7 BiTE and Her2-T7 bi-IgG were evaluated using OVCAR-3 cells and Vy9V52 T cells expanded in vitro. Peripheral blood mononuclear cells (PBMCs) were cultured for seven days in the presence of IL-2 and 0.1 nM T4 IgGto selectively expand the Vy9V52 T cell population. One day before the experiment, OVCAR-3 cells were seeded onto a 96-well plate. On the day of the experiment, varying concentrations of Her2-T7 BiTE or Her2-T7 bi-IgG were added to co-cultures of OVCAR-3 and V / 9V62 T cells at a target-to-effector ratio of 1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and lactate dehydrogenase (LDH) release was quantified using a commercial assay (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity mediated by both Her2-T7 BiTE and Her2- T7 bi-IgG (Figure 1 IB). As a positive control, a Her2 BiTE targeting CD3 was included, while the isotype control (RBD1-T7 BiTE, specific for the SARS-CoV-2 RBD protein) served as a negative control.Example 24 - Cytotoxic evaluation of Her2-T7 BiTE and bi-IgG using expanded Vy9V62 T cells and HeLa cancer cells.

[0404] The cytotoxic activities of Her2-T7 BiTE and Her2-T7 bi-IgG were evaluated using HeLa cells and Vy9V52 T cells expanded in vitro. Peripheral blood mononuclear cells (PBMCs) were cultured for seven days in the presence of IL-2 and 0.1 nM T4 IgGto selectively expand the Vy9V52 T cell population. One day before the experiment, HeLa cells were seeded onto a 96-well plate. On the day of the experiment, varying concentrations of Her2-T7 BiTE or Her2-T7 bi-IgG were added to co-cultures of HeLa and Vy9V52 T cells at a target-to-effector ratio of 1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and lactate dehydrogenase (LDH) release was quantified using a commercial assay (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The resultsdemonstrated robust cytotoxic activity mediated by both Her2-T7 BiTE and Her2-T7 bi-IgG (Figure 11C). As a positive control, a Her2 BiTE targeting CD3 was included, while the isotype control (RBD1-T7 BiTE, specific for the SARS-CoV-2 RBD protein) served as a negative control.Example 25 - Cytotoxic evaluation of Her2-T7 BiTE and bi-IgG using expanded Vy9V62 T cells and PANC-1 cancer cells.

[0405] The cytotoxic activities of Her2-T7 BiTE and Her2-T7 bi-IgG were evaluated using PANC-1 cells and Vy9V52 T cells expanded in vitro. Peripheral blood mononuclear cells (PBMCs) were cultured for seven days in the presence of IL-2 and 0.1 nM T4 IgGto selectively expand the Vy9V52 T cell population. One day before the experiment, PANC-1 cells were seeded onto a 96-well plate. On the day of the experiment, varying concentrations of Her2-T7 BiTE or Her2-T7 bi-IgG were added to co-cultures of PANC-1 and Vy9V52 T cells at a target- to-effector ratio of 1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and lactate dehydrogenase (LDH) release was quantified using a commercial assay (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity mediated by both Her2-T7 BiTE and Her2- T7 bi-IgG (Figure 1 ID). As a positive control, a Her2 BiTE targeting CD3 was included, while the isotype control (RBD1-T7 BiTE, specific for the SARS-CoV-2 RBD protein) served as a negative control.Example 26 - NT5E-T7 BiTE-induced cytotoxicity of enriched Vy9V62 T cells against PC3 cells

[0406] The cytotoxic efficacy of the NT5E-T7 BiTE in combination with Vy9V52 T cells against PC3 cells was evaluated using a lactate dehydrogenase (LDH) release assay. Vy9V52 T cells were enriched from peripheral blood mononuclear cells (PBMCs) utilizing a commercial isolation kit (StemCell, Cat. No. 19255). One day prior to the assay, PC3 cells were seeded into 96-well plates. On the day of the experiment, varying concentrations of NT5E-T7 BiTE were added to co-cultures of PC3 and Vy9V52 T cells at a target-to-effector (T:E) ratio of 1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and LDH release was quantified (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity mediated by the NT5E-T7 BiTE (Figure 8). As a negative control, an RBD1-T7 BiTE, specific for the SARS- CoV-2 RBD protein was used.Example 27 - PRLR-T7 bi-IgG-induced cytotoxicity of enriched Vy9V62 T cells againstMCF-7 cells

[0407] The cytotoxic efficacy of the P2C2-T7 bi-IgG (targeting PRLR) in combination with Vy9V52 T cells against MCF-7 cells was evaluated using a lactate dehydrogenase (LDH) release assay. Vy9V52 T cells were enriched from peripheral blood mononuclear cells (PBMCs) utilizing a commercial isolation kit (StemCell, Cat. No. 19255). One day prior to the assay, MCF-7 cells were seeded into 96-well plates. On the day of the experiment, varying concentrations of P2C2-T7 bi-IgG were added to co-cultures of MCF-7 and Vy9V52 T cells at a target-to-effector (T:E) ratio of 1 :3, followed by incubation for 48 hours at 37°C. After incubation, supernatants were collected and LDH release was quantified (Promega, Cat. No. G1781) according to the manufacturer’s instructions. The results demonstrated robust cytotoxic activity mediated by the P2C2-T7 bi-IgG (Figure 9). As a negative control, an RBD1-T7 bi- IgG, specific for the SARS-CoV-2 RBD protein was used.

[0408] Example 28 - Identification of Antibodies and Antigen Binding Fragments Capable of Binding a Public Domain of y9V62 T cell Receptors.

[0409] The inventors provide strategies to identify antibodies that selectively bind to a public domain motif, such as the TLG sequence, within the gamma delta T cell receptor (TCR). One strategy can utilize phage display with both epitope-focused target engineering and competitive selection. In this process, a panel of recombinant TCR proteins can be engineered for use as selection targets. Across consecutive rounds of panning, the target TCR’s CDR loop can be designed such that the public domain TLG motif remains present and invariant, while the adjacent or flanking residues (e.g., XYZ or ABC, where these represent diverse amino acids) are systematically altered and never repeated. For example, the first round employs a TCR loop sequence “XYZ-TLG-XYZ”, while the second round uses “ABC-TLG-ABC”, and so forth. This can ensure that the only shared epitope among all targets is the TLG motif, guiding the panning process to enrich for antibodies that truly recognize the public domain site independently of its sequence context. To further restrict selection to TLG-specific binders, a competitive binding step can be introduced using a large excess of soluble competitor TCR that shares the same constant region as the target TCRs but lacks the TLG motif in its loop. This competitor protein can be added as a free molecule to the solution during each round, binding and thereby excluding antibodies that recognize either the constant TCR domains or the non- TLG variable regions. Meanwhile, the engineered TCR displaying the TLG motif can beanchored (for example, by biotinylation and immobilization on a streptavidin-coated surface) to permit solid-phase selection, efficient phage binding, and stringent washing to remove weak or non-specific clones. Through four to five rounds, these measures — systematic variation of adjacent residues and the presence of soluble competitor — can greatly enrich for phage clones displaying antibodies with specificity for the TLG public domain motif.

[0410] Another strategy can utilize publicly available and well known computational tools to determine CDRs that can bind to the public domain of the TCR. Using the structure shown in FIG. 2, which shows the crystal structure of HCDR3 of T7 binding to the public domain of the TCR, these tools can be used to determine CDRs, polypeptides loops, or polypeptide domains that can bind to the public domain without undue experimentation.* * *

[0411] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, and those cited elsewhere herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Vyborova, A., Beringer, D. X., Fasci, D., Karaiskaki, F., van Diest, E., Kramer, L., de Haas, A., Sanders, J., Janssen, A., Straetemans, T., Olive, D., Leusen, J., Boutin, L., Nedellec, S., Schwartz, S. L., Wester, M. J., Lidke, K. A., Scotet, E., Lidke, D. S., . . . Kuball, J. (2020). gamma9delta2T cell diversity and the receptor interface with tumor cells. J Clin Invest, 130(9), 4637-4651. https: / / doi.org / 10.1172 / JCI132489

Claims

What is claimed is:

1. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

2. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 having the amino acid sequence of an of a HCDR1, HCDR2, and HCDR3 of a clone of Table 1 and wherein the light chain variable region comprises a LCDR1, LCDR2, and LCDR3 comprising the amino acid sequence of the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone of Table 1.

3. The antibody or antigen-binding fragment of claim 1 or 2, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence that has at least 80% sequence identity to an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone.

4. The antibody or antigen-binding fragment of claim 1 or 2, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequence of an HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are from the same antibody clone.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the heavy chain variable region comprises an amino acid sequence with at least 80% sequence identity to a heavy chain variable region (VH) of an antibody clone of Table 1 and / or the light chain variable region comprises an amino acid sequence with at least 80% sequence identity to the light chain variable region (VL) of the same antibody clone of Table 1.

6. The antibody or antigen-binding fragment of claim 5, wherein the heavy chain variable region comprises the amino acid sequence of a heavy chain variable region of an antibody clone of Table 1 and / or the light chain variable region comprises the amino acid sequence of the same antibody clone of Table 1.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises a full heavy chain with at least 80% sequence identity to a full heavy chain of an antibody clone of Table 1, and comprises a full light chain with at least 80% sequence identity to a full light chain of the same antibody clone of Table 1.

8. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises a full heavy chain of an antibody clone of Table 1, and comprises a full light chain of the same antibody clone of Table 1.

9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody comprises a heavy chain Fab with at least 80% sequence identity to a full heavy chain Fab of an antibody clone of Table 1.

10. The antibody or antigen-binding fragment of claim 9, wherein the heavy chain Fab comprises a full heavy chain Fab of an antibody clone of Table 1.

11. An antibody or antigen-binding fragment comprising a variable region capable of binding a public domain of a Vy9V52 T-cell receptor protein.

12. The antibody or antigen-binding fragment of claim 11, wherein the variable region comprises one, two, three, four, five, or six CDRs capable of binding the public domain of the Vy9V52 T-cell receptor protein.

13. The antibody or antigen-binding fragment of claim 12, wherein one, two, three, four, five, or six of the CDRs comprise an amino acid sequence of VXY, where X is any amino acid.

14. The antibody or antigen-binding fragment of claim 11, wherein the antigen or antigenbinding fragment comprises HCDR3 of clone T7 in Table 1.

15. An antibody or antigen-binding fragment capable of binding a public domain of a Vy9V52 T-cell receptor protein, wherein the antibody or antigen-binding comprises a CDR capable of binding to amino acids TLG located in the public domain of the Vy9V52 T-cell receptor protein, and wherein the CDR comprises an amino acid sequence of VXY, where X is any amino acid.

16. An antibody or antigen-binding fragment capable of binding a public domain of a Vy9V52 T-cell receptor protein, wherein the antibody or antigen-binding comprises a CDR capable of binding to the main chain of the public domain located in the public domain of the Vy9V52 T-cell receptor protein17. The antibody of any one of claims 1 to 16, wherein the antibody is human, chimeric, or humanized.

18. The antibody or antigen-binding fragment of any one of claims 1 to 17, wherein the antibody, or antigen-binding fragment binds a Vy9V52 T-cell receptor protein with a KD of about 10'6M / L to about 10'12M / L.

19. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody is a neutralizing antibody.

20. The antibody or antigen-binding fragment of any one of claims 1 to 19, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody.

21. The antigen-binding fragment of any one of claims 1 to 19, wherein the antigen-binding fragment is a single chain variable fragment (scFv), F(ab’)2, Fab’, Fab, Fv, or rlgG.

22. A bi-specific antibody or antigen-binding fragment comprising two distinct variable regions having a light chain variable region and a heavy chain variable region, wherein one of the two distinct variable region comprises a heavy chain variable region comprising a HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to the HCDR1, HCDR2, HCDR3 from a heavy chain variable region of an antibody clone of Table 1 and a light chain variable region comprising a LCDR1, LCDR2, and LCDR3 having at least 80% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

23. The bi-specific antibody of claim 22, wherein the HCDR1 , HCDR2, and HCDR3 of the heavy chain variable region of the one variable region has at least 95% sequence identity to the HCDR1, HCDR2, HCDR3 from the heavy chain variable region of the antibody clone of Table 1, and wherein the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the one variable region has at least 95% sequence identity to the LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

24. The bi-specific antibody or antigen-binding fragment of claim 22, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the one variable region has at least 100% sequence identity to the HCDR1, HCDR2, HCDR3 from the heavy chain variable region of the antibody clone of Table 1, and wherein the LCDR1, LCDR2, and LCDR3 of the light chain variable region of the one variable region has at least 100% sequence identity to theLCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone of Table 1.

25. The bi-specific antibody or antigen-binding fragment of claim 22, wherein the heavy chain variable region of the one variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 80% sequence identity to SEQ ID NOs: 12, 22, and 57, respectively, and wherein the light chain variable region of the one variable region comprises a LCRD1, LCDR2, and LCDR3 having at least 80% sequence identity to SEQ ID NOs: 4, 5, and 58, respectively.

26. The bi-specific antibody or antigen-binding fragment of claim 22, wherein the heavy chain variable region of the one variable region comprises a HCDR1, HCDR2, and HCDR3 having at least 95% sequence identity to SEQ ID NOs: 12, 22, and 57, respectively, and wherein the light chain variable region of the one variable region comprises a LCRD1, LCDR2, and LCDR3 having at least 95% sequence identity to SEQ ID NOs: 4, 5, and 58, respectively.

27. The bi-specific antibody or antigen-binding fragment of claim 22, wherein the heavy chain variable region of the one variable region comprises a HCDR1, HCDR2, and HCDR3 comprising SEQ ID NOs: 12, 22, and 57, respectively, and wherein the light chain variable region of the one variable region comprises a LCRD1, LCDR2, and LCDR3 comprising SEQ ID NOs: 4, 5, and 58, respectively.

28. The bi-specific antibody or antigen-binding fragment of any one of claims 22 to 27, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region that are able to bind to a tumor cell.

29. The bi-specific antibody or antigen-binding fragment of any one of claims 22 to 28, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region that are able to bind to an analyte.

30. The bi-specific antibody or antigen-binding fragment of any one of claims 22 to 29, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region that are able to bind to a tumor antigen.

31. The bi-specific antibody or antigen-binding fragment of any one of claims 22 to 30, wherein the other variable region of the two distinct variable regions comprises a heavy chain variable region and a light chain variable region from atezolizumab, belantamab, bevacizumab, blinatumomab, brentuximab, cetuximab, daratumumab, dinutuximab, elotuzumab, enfortumab, gemtuzumab, ibritumomab, inotuzumab, necitumumab, obinutuzumab,ofatumumab, olaratumab, panitumumab, pertuzumab, ramucirumab, rituximab, sacituzumab, or trastuzumab.

32. The bi-specific antibody of any one of claims 22 to 31, wherein the antibody is human, chimeric, or humanized.

33. The bi-specific antibody or antigen-binding fragment of any one of claims 22 to 32, wherein the antibody, or antigen-binding fragment binds a Vy9V52 T-cell receptor protein with a KD of about 10'6M / L to about 10'12M / L.

34. The bi-specific antibody of any one of claims 22 to 33, wherein the antibody is a bi- specific T cell engager antibody.

35. The bi-specific antibody of any one of claims 22 to 34, wherein the antibody is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, or a monoclonal antibody.

36. A polypeptide comprising the antigen-binding fragment of any one of claims 1 to 35.

37. The polypeptide of claim 36, wherein the polypeptide comprises at least two antigenbinding fragments, wherein each antigen-binding fragment is independently selected from an antigen-binding fragment of any one of claims 1 to 21.

38. The polypeptide of claim 36 or 37, wherein the polypeptide is multivalent.

39. The polypeptide of any one of claims 36 to 38, wherein the polypeptide is bispecific.

40. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 35 or the polypeptide of any one of claims 36 to 39.

41. The composition of claim 40, wherein the composition comprises a pharmaceutical excipient.

42. The composition of claim 40 or 41, wherein the composition further comprises an adjuvant.

43. The composition of any one of claims 40 to 42, wherein the composition is formulated for parenteral, intravenous, subcutaneous, intramuscular, or intranasal administration.

44. The composition of any one of claims 1 to 43, wherein the composition comprises at least two antibodies or antigen-binding fragments.

45. One or more nucleic acids encoding the antibody or antigen-binding fragment of any one of claims 1 to 20 or the polypeptide of claim 39.

46. A vector comprising the nucleic acid(s) of claim 45.

47. A host cell comprising the nucleic acid of claim 45, or the vector of claim 26.

48. The host cell of claim 47, wherein the host cell is a human cell, B cell, T cell, Chinese hamster ovary, NSO murine myeloma cell, or PER.C6 cell.

49. A method of a making a cell comprising transferring the nucleic acid(s) of claim 45 or the vector of claim 46 into a cell.

50. The method of claim 49, wherein the method further comprises culturing the cell under conditions that allow for expression of a polypeptide from the nucleic acid.

51. The method of claim 50, wherein the method further comprising isolating the expressed polypeptide.

52. The method of any one of claims 49 to 51, wherein the cell is a human cell, B cell, T cell, Chinese hamster ovary, NSO murine myeloma cell, or PER.C6 cell.

53. A method for producing a polypeptide comprising transferring the nucleic acid(s) of claim 45 or the vector of claim 46 into a cell and isolating polypeptides expressed from the nucleic acid.

54. The method of claim 53, wherein the cell is a human cell, B cell, T cell, Chinese hamster ovary, NSO murine myeloma cell, or PER.C6 cell.

55. A method for treating or preventing cancer in a patient, the method comprising administering to the patient the antibody or antigen-binding fragment of any one of claims 1 to 35, the polypeptide of any one of claims 35 to 39, the composition of any one of claims 40 to 44, or the host cell of claim 47 or 48.

56. The method of claim 55, wherein the patient is a human patient.

57. The method of claim 55 or 56, wherein the patient has one or more symptoms of cancer.

58. The method of claim 55 or 56, wherein the patient does not have any symptoms of cancer.

59. The method of any one of claims 55 to 58, wherein the patient has been diagnosed with cancer.

60. The method of any one of claims 55 to 58, wherein the patient has not been diagnosed with cancer.

61. The method of any one of claims 55 to 60, wherein the patient has been previously treated for cancer.

62. The method of any one of claims 55 to 61, wherein the patient is administered an additional therapy.

63. The method of claim 62, wherein the additional therapy comprises radiotherapy, chemotherapy, or immunotherapy.

64. A method for treating or preventing a disease indicated for an immunotherapy in a patient, the method comprising administering to the patient the antibody or antigen-binding fragment of any one of claims 1 to 35, the polypeptide of any one of claims 35 to 39, the composition of any one of claims 40 to 44, or the host cell of claim 47 or 48.

65. The method of claim 64, wherein the patient is a human patient.

66. The method of claim 64 or 65, wherein the patient has one or more symptoms of the disease.

67. The method of claim 64 or 65, wherein the patient does not have one or more symptoms of the disease.

68. The method of claim 64 or 65, wherein the patient has been diagnosed with the disease.

69. The method of claim 64 or 65, wherein the patient has not been diagnosed with the disease.

70. The method of any of claims 64 to 69, wherein the patient has been previously treated for the disease.

71. The method of claim 70, wherein the patient was resistant to the previous treatment.

72. A method for evaluating a sample from a patient, the method comprising contacting a biological sample from the patient, or extract thereof, with at least one antibody, antigenbinding fragment, or polypeptide of any one of claims 1 to 39.

73. The method of claim 72, wherein the at least one antibody, antigen-binding fragment, or polypeptide is operatively linked to a detectable label.

74. The method of claim 72 or 73, wherein the method further comprises incubating the antibody, antigen-binding fragment, or polypeptide under conditions that allow for the binding of the antibody, antigen-binding fragment, or polypeptide to antigens in the biological sample or extract thereof.

75. The method of any one of claims 72 to 74, wherein the method further comprises detecting the binding of an antigen to the antibody, antigen-binding fragment, or polypeptide.

76. The method of any one of claims 72 to 75, wherein the method further comprises contacting the biological sample with at least one capture antibody, antigen, or polypeptide.

77. The method of claim 76, wherein the at least one capture antibody, antigen-binding fragment, or polypeptide comprises at least one antibody or antigen-binding fragment of claims 1 to 20.

78. The method of claim 76 or 77, wherein the capture antibody or fragment is linked to a solid support.

79. The method of any one of claims 72 to 78, wherein the biological sample comprises a tissue sample or a blood sample.

80. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T1 in Table 1.

81. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T2 in Table 1.

82. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T3 in Table 1.

83. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T4 in Table 1.

84. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T5 in Table 1.

85. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T6 in Table 1.

86. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7 in Table 1.

87. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.2 in Table 1.

88. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.3 in Table 1.

89. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.4 in Table 1.

90. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.5 in Table 1.

91. An antibody or antigen-binding fragment comprising an LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of clone T7.6 in Table 1.

92. An antibody or antigen-binding fragment comprising SEQ ID NOs: 82-85.

93. An antibody or antigen-binding fragment comprising SEQ ID NOs: 84, 85, 90, and 91.

94. An antibody or antigen-binding fragment comprising SEQ ID NOs: 84, 85, 98, and 99.

95. A method of treating a patient, the method comprising administering the antibody of any one of claims 80 to 94 to the patient.

96. A method of treating a patient, the method comprising administering a pharmaceutical composition comprising the antibody of any one of claims 80 to 94 to the patient.

97. A method of engaging a T cell with a cancer cell, the method comprising contacting the T cell and the cancer cell with the antibody or antigen-binding fragment of any one of claims 22 to 35.

98. The method of claim 97, wherein the T cell is a y952 T cell.

99. The method of claim 97 or 98, wherein the antibody or antigen-binding fragment comprises a variable region that is able to bind to an antigen expressed on the cancer cell.

100. The method of claim 99, wherein the antigen expressed on the cancer cell is a tumor antigen.

101. The method of any one of claims 97 to 100, wherein the contacting occurs in vivo.

102. The method of any one of claims 97 to 100, wherein the contacting occurs ex vivo.

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