Engineered T cell receptors and methods of use

Abstract

The present disclosure provides engineered T cell receptors (TCRs), cells containing TCRs, and methods for producing and using TCRs. The present disclosure relates to TCRs that specifically recognize epitopes derived from tumor antigen COL6A3. Accordingly, aspects of the present disclosure relate to engineered T cell receptors (TCRs), nucleic acids encoding TCRs, and cells containing the nucleic acids and TCRs. Also provided are compositions comprising the cells, nucleic acids, or engineered TCRs of the present disclosure, methods for producing the cells, and methods for using aspects of the present disclosure for therapeutic treatment.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 036,539, filed June 9, 2020, which is incorporated herein by reference in its entirety.

[0002] I. FIELD OF THE INVENTION The present invention relates to the field of cancer therapy. [Background technology]

[0003] II. Background Adoptive T cell therapy is a potentially powerful cancer treatment that genetically modifies natural T cells to make them tumor-specific and improve their ability to destroy tumor cells. Genetically modified T cells can express chimeric antigen receptors (CARs) or T cell receptors (TCRs) and have shown impressive results in multiple clinical trials. TCR-engineered T (TCR-T) cells show great promise against tumors. The efficacy of TCRs depends on their interaction with the peptide-major histocompatibility complex (pMHC), a complex formed by peptides bound to MHC. Intracellular antigens are cleaved into peptide chains and presented by MHC molecules to form pMHC. Cytoplasmic proteins expressed by class I MHC proteins, most of which are defective ribosomal translation products, are proteolytically cleaved into peptide chains. These peptides then bind to class I MHC proteins, which are expressed on the cell surface of all nucleated cells. Some cells, called antigen-presenting cells (APCs), express class II MHC proteins. They internalize foreign proteins by endocytosis, cleave them into peptide chains, and bind to T cell-derived class II MHC proteins, T cell receptors, which recognize these pMHC and cause cancer cell death. (Human class I MHC proteins are expressed from three gene regions: HLA-A, HLA-B, and HLA-C, while human class II MHC proteins are also expressed from three gene regions: HLA-DR, HLA-DP, and HLA-DQ.) Engineering TCRs that are directed against cancer-specific antigens and useful for cancer treatment is needed. Summary of the Invention

[0004] The present disclosure provides engineered T cell receptors (TCRs), cells containing TCRs, and methods of making and using TCRs. The present disclosure relates to TCRs that specifically recognize the epitope FLLDGSANV (SEQ ID NO:15) from the tumor antigen COL6A3. Thus, aspects of the present disclosure include: Another aspect relates to a polypeptide comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000001.tif4128. The present invention relates to a polypeptide comprising an antigen-binding variable region including a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000002.tif4128.

[0005] The present disclosure also provides T cell receptors (TCRs) and engineered TCRs, including TCR-a polypeptides and TCR-b polypeptides, for example, wherein the TCR-a polypeptide is and a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000003.tif4128, The present invention provides a TCR, etc., comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000004.tif4128.

[0006] Further aspects relate to nucleic acids encoding the polypeptides and engineered TCRs, nucleic acid vectors comprising one or more nucleic acids of the present disclosure, and cells comprising the polypeptides, engineered TCRs and / or nucleic acids of the present disclosure. Compositions comprising the polypeptides, cells, nucleic acids, or engineered TCRs of the present disclosure are also provided. Further aspects relate to methods for making engineered cells, comprising introducing the nucleic acids or vectors of the present disclosure into cells. Further aspects relate to methods for treating cancer in a subject, comprising administering to a subject in need thereof a polypeptide, composition, cell, nucleic acid, or engineered TCR. Methods include methods for reducing tumor burden; dissolving cancer cells; killing tumors / cancer cells; increasing overall survival; reducing the risk of developing cancer or developing a tumor; increasing recurrence-free survival; preventing cancer; and / or reducing, eliminating, or decreasing the spread or metastasis of cancer, comprising administering to a subject in need thereof a polypeptide, composition, cell, nucleic acid, or engineered TCR.

[0007] A further aspect relates to a fusion protein comprising the TCR and CD3 binding region of the present disclosure. The CD3 binding region can comprise a CD3-specific fragment antigen-binding (Fab), a single-chain variable fragment (scFv), a single-domain antibody, or a single-chain antibody. Exemplary CD3-specific fragment antigen-binding (Fab) fragments are known in the art. For example, US20180222981, incorporated herein by reference, discloses a variable region that specifically binds to CD3 and can be used in aspects of the present disclosure. Anti-CD3 antibodies and variable regions are disclosed in US20180117152, also incorporated by reference.

[0008] A polypeptide or TCR-a polypeptide of the disclosure may comprise a CDR3 comprising an amino acid sequence that has 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 range derivable therein) sequence identity to SEQ ID NO:8, or that has at least 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 range derivable therein) sequence identity to SEQ ID NO:8. A polypeptide may comprise a CDR3 that comprises the amino acid sequence of SEQ ID NO:8. The polypeptide or TCR-b polypeptide of the present disclosure may be 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to TIFF0007827645000005.tif4128, or exactly 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 range derivable therein) sequence identity to TIFF0007827645000005.tif4128. A polypeptide or TCR-b polypeptide of the present disclosure may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO: 14. An engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO: 8 and a TCR-b polypeptide comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 14.

[0009] The polypeptide may comprise a variable region comprising CDR1, CDR2, and CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having at least 80% sequence identity to TSGFNG (SEQ ID NO:6). In some aspects, the variable region comprises a CDR1 having 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 range derivable therein) sequence identity to SEQ ID NO:6, or at least 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 range derivable therein) sequence identity to SEQ ID NO:6. In some aspects, the variable region comprises a CDR2 having at least 80% sequence identity to NVLDGL (SEQ ID NO:7). The variable region may comprise a CDR2 that has 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 range derivable therein) sequence identity to SEQ ID NO:7, or that has at least 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 range derivable therein) sequence identity to SEQ ID NO:7. The variable region may comprise a CDR1 that comprises the amino acid sequence of SEQ ID NO:6 and / or a CDR2 that comprises the amino acid sequence of SEQ ID NO:7. The variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4.In some aspects, the variable region comprises an amino acid sequence that has 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 range derivable therein) sequence identity to, or has at least 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 range derivable therein) sequence identity to SEQ ID NO:4. In some aspects, the variable region comprises the amino acid sequence of SEQ ID NO:4. In some aspects, the polypeptide comprises a T cell receptor alpha (TCR-a) variable region. In some aspects, the polypeptide comprises a TCR-a variable region and a constant region. The polypeptide may further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:5. In some aspects, the signal peptide comprises an amino acid sequence that has 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 range derivable therein) sequence identity to, or has at least 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 range derivable therein) sequence identity to SEQ ID NO:5. In some aspects, the signal peptide comprises the amino acid sequence of SEQ ID NO:5. In some aspects, the variable region comprises CDR1, CDR2 and / or CDR3.

[0010] The variable region may comprise a CDR1 having at least 80% sequence identity to SEQ ID NO:12. In some aspects, the variable region comprises a CDR1 having 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 range derivable therein) sequence identity to SEQ ID NO:12, or at least 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 range derivable therein) sequence identity to SEQ ID NO:12. The variable region may comprise a CDR2 having at least 80% sequence identity to YYNGEE (SEQ ID NO:13). In some aspects, the variable region comprises a CDR2 that has 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 range derivable therein) sequence identity to SEQ ID NO:13, or at least 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 range derivable therein) sequence identity to SEQ ID NO:13. The variable region may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 and / or a CDR2 comprising the amino acid sequence of SEQ ID NO: 13. In some aspects, the variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10.In some aspects, the variable region comprises an amino acid sequence that has 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 range derivable therein) sequence identity to, or has at least 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 range derivable therein) sequence identity to SEQ ID NO: 10. In some aspects, the variable region comprises the amino acid sequence of SEQ ID NO: 10. The polypeptide may comprise a T cell receptor beta (TCR-b) variable region. In some aspects, the polypeptide comprises a TCR-b variable region and a constant region. The polypeptide may comprise, or further comprise, a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:11. In some aspects, the signal peptide comprises an amino acid sequence that has 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 range derivable therein) sequence identity to, or has at least 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 range derivable therein) sequence identity to SEQ ID NO:11. The signal peptide comprises the amino acid sequence of SEQ ID NO:11. In some aspects, the variable region can comprise CDR1, CDR2 and / or CDR3.

[0011] The TCR may comprise a TCR-a polypeptide comprising a variable region comprising CDR1, CDR2, and CDR3, and a TCR-b polypeptide comprising a variable region comprising CDR1, CDR2, and CDR3. The TCR-a polypeptide may comprise a CDR1 having at least 80% sequence identity to SEQ ID NO:6, and / or the TCR-b polypeptide may comprise a CDR1 having at least 80% sequence identity to SEQ ID NO:12. The TCR-a polypeptide may comprise a CDR1 having at least 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 range derivable therein) sequence identity to SEQ ID NO:6, and / or the TCR-b polypeptide may comprise a CDR1 having at least 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 range derivable therein) sequence identity to SEQ ID NO:12. The TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:6, and the TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:12. In some aspects, the TCR-a polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:13.In some aspects, the TCR-a polypeptide comprises a CDR2 having 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 range derivable therein) sequence identity to SEQ ID NO:7, or at least 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 range derivable therein) sequence identity to SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 having 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 range derivable therein) sequence identity to SEQ ID NO: or 100% (or any range derivable therein) sequence identity to NO:13, or comprises a CDR2 having at least 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 range derivable therein) sequence identity. In some aspects, the TCR-a polypeptide comprises a CDR2 comprising the amino acid sequence of SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 comprising the amino acid sequence of SEQ ID NO:13. The CDR1, CDR2, and CDR3 of the TCR-a polypeptide may comprise the amino acid sequences of SEQ ID NOs:6, 7, and 8, respectively, and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide may comprise the amino acid sequences of SEQ ID NOs:12, 13, and 14, respectively. In some aspects, the TCR-a variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, and the TCR-b variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10.In some aspects, the TCR-a variable region comprises an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO:4, or at least 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 range derivable therein) sequence identity to SEQ ID NO:4, and the TCR-b variable region comprises an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO:4. or 100% (or any range derivable therein) sequence identity to NO:10, or at least 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 range derivable therein) sequence identity to NO:10. In some aspects, the TCR-a polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, and the TCR-b polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:9.In some aspects, the TCR-a polypeptide comprises an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO:3, or at least 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 range derivable therein) sequence identity to SEQ ID NO:3, and the TCR-b polypeptide comprises an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO: The TCR-a and / or TCR-b polypeptides may comprise an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO:9, or having at least 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 range derivable therein) sequence identity to SEQ ID NO:9. The TCR-a and / or TCR-b polypeptides may comprise a signal peptide. The signal peptide may comprise SEQ ID NO:5 or 11. In some aspects, the signal peptide comprises an amino acid sequence that has 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 range derivable therein) sequence identity to SEQ ID NO:5 or 11, or has at least 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 range derivable therein) sequence identity.

[0012] The TCR may contain modifications or be chimeric. The variable region of the TCR may be fused to a TCR constant region that is different from the constant region of the cloned TCR that specifically binds the peptide of the present disclosure.

[0013] The TCR-a polypeptide and the TCR-b polypeptide may be operably linked. The term "operably linked" can refer to a covalent bond, such as a peptide bond (e.g., two elements are polypeptides and are on the same polypeptide), or a non-covalent bond, such as van der Waals forces (e.g., two polypeptides that have some degree of specific binding affinity for each other). The TCR-a polypeptide and the TCR-b polypeptide are operably linked through a peptide bond. The TCR-a polypeptide and the TCR-b polypeptide are on the same polypeptide, with the TCR-b being amino-proximal to the TCR-a. The polypeptide may be further defined as a single-chain TCR. The TCR-a polypeptide and the TCR-b polypeptide may be on the same polypeptide, with the TCR-a being amino-proximal to the TCR-b. The TCR may include a linker between the TCR-a polypeptide and the TCR-b polypeptide. The linker may include glycine and serine residues. Additional linkers useful in the polypeptides and TCRs of the present disclosure are described herein. When a first region is attached to the carboxy terminus of a second region, the first region is carboxy-proximal to the second region. There may be additional intervening amino acid residues between the first and second regions. Thus, unless specifically specified as having no intervening amino acid residues, these regions need not be directly adjacent. The term "amino-proximal" is similarly defined as when a first region is attached to the amino terminus of the second region, the first region is amino-proximal to the second region. Similarly, unless otherwise specified, there may be additional intervening amino acid residues between the first and second regions.

[0014] CDRs may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23 or more (or any range derivable therein) contiguous amino acid residues flanking either or both sides of a particular CDR sequence; thus, there may be one or more additional amino acids at the N-terminus or C-terminus of a particular CDR sequence, such as those set forth in SEQ ID NOs:6-8 and 12-14.

[0015] The TCR or fusion protein can be conjugated to a detection or therapeutic agent. The agent can include a fluorescent molecule, a radioactive molecule, or a toxin. In some aspects, the TCR or fusion protein is conjugated to an agent described herein.

[0016] The present disclosure also provides: a TCR-α polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000006.tif4128; and / or A nucleic acid encoding a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000007.tif4128 is provided. In some aspects, the nucleic acid comprises a TCR-a polypeptide comprising a CDR3 comprising an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO:8, or at least 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 range derivable therein) sequence identity to SEQ ID NO:8. or 100% (or any range derivable therein) sequence identity to NO:14, or encodes a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 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 range derivable therein) sequence identity. The nucleic acid may encode a TCR-a polypeptide comprising CDR1, CDR2 and CDR3, and / or a TCR-b polypeptide comprising CDR1, CDR2 and CDR3. The nucleic acid may encode a TCR-a comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:6 and / or a TCR-b comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:12.In some aspects, the nucleic acid comprises a TCR-a and / or a CDR1 having 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 range derivable therein) sequence identity to SEQ ID NO:6, or at least 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 range derivable therein) sequence identity to SEQ ID NO:6. or 100% (or any range derivable therein) sequence identity to NO:12, or encodes a TCR-b comprising a CDR1 having at least 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 range derivable therein) sequence identity to NO:12. In some aspects, the nucleic acid encodes a TCR-a comprising a CDR2 having at least 80% sequence identity to SEQ ID NO:7 and / or a TCR-b comprising a CDR2 having at least 80% sequence identity to SEQ ID NO:13. In some aspects, the nucleic acid encodes a TCR-a comprising a CDR2 having at least 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 range derivable therein) sequence identity to SEQ ID NO:7 and / or a TCR-b comprising a CDR2 having at least 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 range derivable therein) sequence identity to SEQ ID NO:13.In some aspects, the nucleic acid encodes a TCR-a variable region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4 and / or a TCR-b variable region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10. In some aspects, the nucleic acid comprises a TCR-a variable region comprising an amino acid sequence having 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 range derivable therein) sequence identity to SEQ ID NO:4, or at least 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 range derivable therein) sequence identity to SEQ ID NO:4. or encodes a TCR-b variable region comprising an amino acid sequence having 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 range derivable therein) sequence identity to NO:10, or at least 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 range derivable therein) sequence identity. In some aspects, the nucleic acid encodes a TCR-a polypeptide chain comprising the amino acid sequence of SEQ ID NO:4 and / or a TCR-b chain comprising the amino acid sequence of SEQ ID NO:10. In some aspects, the nucleic acid comprises SEQ ID NO:1 and / or SEQ ID NO:2.In some aspects, the nucleic acid has 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 range derivable therein) sequence identity to SEQ ID NO:1, or at least 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 range derivable therein) sequence identity to SEQ ID NO:1, and / or 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:2, or at least 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 range derivable therein) sequence identity to NO:2.

[0017] The nucleic acid may comprise a TCR-a (TRA) and a TCR-b (TRB) gene. The nucleic acid may be polycistronic. The nucleic acid may also comprise an internal ribosome entry site (IRES) or a P2A linker. The nucleic acid may comprise a cDNA encoding a TCR-a and / or a TCR-b gene. The nucleic acid may encode or further encode a polypeptide comprising a CD3-binding region. In some aspects, the CD3-binding region comprises a CD3-specific fragment antigen-binding (Fab), a single-chain variable fragment (scFv), a single-domain antibody, or a single-chain antibody.

[0018] The vector may contain both TCR-a and TCR-b genes. The vector may contain a promoter that drives expression of the nucleic acid. The promoter may include a murine stem cell virus (MSCV) promoter. In some aspects, the cells include stem cells, progenitor cells, immune cells, or natural killer (NK) cells. In some aspects, the cells include hematopoietic stem or progenitor cells, T cells, cells differentiated from mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs). The cells may be isolated from or derived from peripheral blood mononuclear cells (PBMCs). In some aspects, the T cells include cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, invariant NK T (iNKT) cells, gamma-delta T cells, NKT cells, or regulatory T cells. In some aspects, the cells are isolated from a cancer patient. In some aspects, the cells are isolated from a non-cancer patient. In some aspects, the cells are isolated from a healthy patient. The cells may be frozen or not. The cells may be in cell culture. In some aspects, the cells lack endogenous expression of TCR genes. In some aspects, the cells further comprise a chimeric antigen receptor (CAR).

[0019] In some aspects, the composition is determined to be serum-free, mycoplasma-free, endotoxin-free, and / or sterile. The method may include, or may further include, culturing the cells in a medium, incubating the cells under conditions that allow the cells to divide, screening the cells, and / or freezing the cells.

[0020] In some aspects, the subject has been diagnosed with cancer, such as a cancer described herein. The cancer may include a solid tumor. The subject may have previously received cancer treatment. The subject may be determined to be resistant to a previous treatment. The method may include, or may further include, administration of an additional therapeutic agent. The cancer may include glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, glioblastoma, cervical cancer, endometrial cancer, ovarian cancer, or melanoma cancer. The cancer may include kidney cancer. The cancer may include glioblastoma. The cancer may include COL6A3+ cancer. The subject or patient may be determined to have COL6A3+ cancer cells or cancer cells positive for the epitope of SEQ ID NO: 15. In some aspects, the cancer is a COL6A3+ overexpressing cancer, such as one that is overexpressed compared to non-cancerous subjects. The subject or patient may be a subject determined to have COL6A3 overexpression in a biological sample. The biological sample may include cancer cells or a biopsy. The subject may be a mammal. In some aspects, the subject includes an experimental animal such as a mouse, rat, rabbit, dog, cat, horse, or pig. In some aspects, the subject is a human. In some aspects, the subject has been determined to be HLA-A2 and / or HLA-A*0201 positive.

[0021] The composition of the present disclosure can be formulated as a vaccine.The composition and method of the present disclosure provide a preventive therapy for preventing cancer.The composition and method of the present disclosure provide a therapeutic therapy for treating existing cancer, such as for treating patients with cancer.The composition can comprise or further comprise an adjuvant.Adjuvants are known in the art and include, for example, TLR agonists and aluminum salts.

[0022] The methods of the present disclosure may include, or may further include, screening the cells for one or more cellular properties, such as for TCR expression, incorporation of a nucleic acid encoding a TCR gene, or for immunogenic properties, such as binding of the TCR to a cancer antigen, such as COL6A3 or a peptide of SEQ ID NO:15.

[0023] The method may include, or may further include, administering cells or a composition comprising the cells, wherein the cells comprise autologous cells. In some aspects, the cells comprise non-autologous cells. The cells may be allogeneic or xenogeneic.

[0024] Treating or treating can refer to any treatment of a disease in a mammal, including (i) preventing the disease, i.e., preventing the onset of clinical symptoms of the disease by administration of a protective composition prior to the induction of the disease; (ii) inhibiting the disease, i.e., preventing the onset of clinical symptoms of the disease by administration of a protective composition after an inducing event but prior to the clinical appearance or reappearance of the disease; (iii) arresting the disease, i.e., preventing the onset of clinical symptoms by administration of a protective composition after their initial appearance; and / or (iv) alleviating the disease, i.e., causing regression of clinical symptoms by administration of a protective composition after their initial appearance. In some aspects, treating can exclude prevention of the disease.

[0025] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value.

[0026] When used in conjunction with the term "comprising," the use of the words "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0027] As used herein, the terms "or" and "and / or" are utilized to describe two or more elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.

[0028] 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"), "characterized by" (and any form of including, such as "characterized as") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude further, unrecited elements or method steps.

[0029] Compositions and methods for their use can "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this application. The phrase "consisting of" excludes any unspecified elements, steps, or components. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term "comprising" can also be implemented in the context of the term "consisting of" or "essentially consisting of."

[0030] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as "use of" any compound, composition, or agent discussed herein to achieve or carry out the stated therapeutic, diagnostic, or physiological purpose or effect.

[0031] The use of one or more sequences or compositions can be utilized according to any of the methods described herein. Other aspects are discussed throughout this application. Any aspect or aspect discussed with respect to one aspect of this disclosure applies to other aspects of this disclosure as well, and vice versa.

[0032] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to make or utilize any composition of the invention. Aspects of an embodiment described in an example are also embodiments that can be implemented in the context of embodiments discussed elsewhere in a different example or elsewhere in this application, for example, in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legends.

[0033] [The present invention 1001] TIFF0007827645000008.tif4128 A polypeptide comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to [The present invention 1002] 1001. The polypeptide of the present invention, wherein CDR3 comprises the amino acid sequence of SEQ ID NO:8. [The present invention 1003] The polypeptide of claim 1001 or 1002, wherein the variable region comprises CDR1, CDR2, and / or CDR3. [The present invention 1004] 1003. The polypeptide of the invention, wherein the variable region comprises a CDR1 having at least 80% sequence identity to TSGFNG (SEQ ID NO:6). [The present invention 1005] The polypeptide of claim 1003 or 1004, wherein the variable region comprises a CDR2 having at least 80% sequence identity to NVLDGL (SEQ ID NO:7). [The present invention 1006] The polypeptide of the present invention 1004 or 1005, wherein the variable region comprises CDR1 comprising the amino acid sequence of SEQ ID NO:6 and / or CDR2 comprising the amino acid sequence of SEQ ID NO:7. [The present invention 1007] The polypeptide of any of claims 1001 to 1006, wherein the variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4. [The present invention 1008] 1007. The polypeptide of the present invention, wherein the variable region comprises the amino acid sequence of SEQ ID NO:4. [The present invention 1009] Any of the polypeptides of 1001 to 1008 of the present invention, comprising a T cell receptor alpha (TCR-a) variable region. [The present invention 1010] 1009. A polypeptide of the present invention comprising a TCR-α variable region and a constant region. [The present invention 1011] The polypeptide of any one of 1001 to 1010 of the present invention, further comprising a signal peptide. [The present invention 1012] 1011. The polypeptide of the invention, wherein the signal peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:5. [The present invention 1013] 1012. The polypeptide of the present invention, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:5. [The present invention 1014] TIFF0007827645000009.tif4128 A polypeptide comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to [The present invention 1015] The polypeptide of the present invention, wherein CDR3 comprises the amino acid sequence of SEQ ID NO:14. [The present invention 1016] The polypeptide of the invention 1014 or 1015, wherein the variable region comprises CDR1, CDR2, and / or CDR3. [The present invention 1017] 1016. The polypeptide of the invention, wherein the variable region comprises a CDR1 having at least 80% sequence identity to SGDLS (SEQ ID NO:12). [The present invention 1018] The polypeptide of the invention 1016 or 1017, wherein the variable region comprises a CDR2 having at least 80% sequence identity to YYNGEE (SEQ ID NO:13). [The present invention 1019] The polypeptide of the present invention 1017 or 1018, wherein the variable region comprises CDR1 comprising the amino acid sequence of SEQ ID NO:12 and / or CDR2 comprising the amino acid sequence of SEQ ID NO:13. [The present invention 1020] The polypeptide of any of claims 1014 to 1019, wherein the variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10. [The present invention 1021] The polypeptide of the present invention 1020, wherein the variable region comprises the amino acid sequence of SEQ ID NO:10. [The present invention 1022] Any of the polypeptides of 1014 to 1021 of the present invention, which comprises a T cell receptor beta (TCR-b) variable region. [The present invention 1023] A polypeptide of the present invention 1022 comprising a TCR-b variable region and a constant region. [The present invention 1024] The polypeptide of any one of 1014 to 1023 of the present invention, further comprising a signal peptide. [The present invention 1025] 1024. The polypeptide of the invention, wherein the signal peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11. [The present invention 1026] 1025. The polypeptide of the invention, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:11. [The present invention 1027] A nucleic acid encoding any one of the polypeptides of the present invention 1001 to 1026. [The present invention 1028] 1. An engineered T cell receptor (TCR) comprising a TCR-a polypeptide and a TCR-b polypeptide, TCR-α polypeptide, TIFF0007827645000010.tif4128 and a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TCR-b polypeptide, TIFF0007827645000011.tif4128 and a CDR3 comprising an amino acid sequence having at least 80% sequence identity to Engineered TCR. [The present invention 1029] 1028. The TCR of the present invention, wherein the TCR-a polypeptide comprises a CDR3 comprising the amino acid sequence of SEQ ID NO:8, and the TCR-b polypeptide comprises a CDR3 comprising the amino acid sequence of SEQ ID NO:14. [The present invention 1030] A TCR of the present invention 1028 or 1029, comprising a TCR-a polypeptide comprising a variable region comprising CDR1, CDR2, and CDR3, and a TCR-b polypeptide comprising a variable region comprising CDR1, CDR2, and CDR3. [The present invention 1031] A TCR of the present invention 1030, wherein the TCR-a polypeptide comprises a CDR1 having at least 80% sequence identity to SEQ ID NO:6 and / or the TCR-b polypeptide comprises a CDR1 having at least 80% sequence identity to SEQ ID NO:12. [The present invention 1032] A TCR of the present invention 1031, wherein the TCR-a polypeptide comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:6, and the TCR-b polypeptide comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:12. [The present invention 1033] A TCR according to any one of claims 1030 to 1032, wherein the TCR-a polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:13. [The present invention 1034] The TCR of the present invention 1033, wherein the TCR-a polypeptide comprises a CDR2 comprising the amino acid sequence of SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 comprising the amino acid sequence of SEQ ID NO:13. [This invention 1035] A TCR of any of the present inventions 1030 to 1034, wherein CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively. [The present invention 1036] A TCR of any of claims 1030 to 1035, wherein the TCR-a polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, and the TCR-b polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10. [This invention 1037] 1036. A TCR of the present invention, wherein the TCR-a polypeptide comprises the amino acid sequence of SEQ ID NO:4 and the TCR-b polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10. [The present invention 1038] Any of TCRs 1028 to 1037 of the present invention, which contains modifications or is chimeric. [This invention 1039] Any of the TCRs of the present invention 1028 to 1038, in which the TCR-a polypeptide and the TCR-b polypeptide are functionally linked. [The present invention 1040] 1039. The TCR of the present invention, wherein the TCR-a polypeptide and the TCR-b polypeptide are operably linked via a peptide bond. [This invention 1041] The TCR of the present invention 1039, which is a single-chain TCR. [The present invention 1042] A TCR of the invention 1040 or 1042, wherein the TCR-a polypeptide and the TCR-b polypeptide are on the same polypeptide and the TCR-b is amino proximal to the TCR-a. [This invention 1043] A TCR of the invention 1040 or 1042, wherein the TCR-a polypeptide and the TCR-b polypeptide are on the same polypeptide and the TCR-a is amino-proximal to the TCR-b. [This invention 1044] Any of the TCRs of 1040 to 1043 of the present invention, which comprises a linker between the TCR-a polypeptide and the TCR-b polypeptide. [This invention 1045] The TCR of the present invention, 1040 to 1044, wherein the linker contains glycine and serine residues. [The present invention 1046] A fusion protein comprising any one of the TCRs of the present invention 1028 to 1045 and a CD3-binding region. [This invention 1047] The fusion protein of the present invention 1046, wherein the CD3 binding region comprises a CD3-specific fragment antigen binding (Fab), a single-chain variable fragment (scFv), a single domain antibody, or a single-chain antibody. [This invention 1048] The TCR of any one of claims 1028 to 1045 or the fusion protein of claim 1046 or 1047, wherein the TCR or fusion protein is conjugated to a detection agent or a therapeutic agent. [This invention 1049] 1048. The TCR or fusion protein of the present invention, wherein the agent comprises a fluorescent molecule, a radioactive molecule, or a toxin. [The present invention 1050] TIFF0007827645000012.tif4128 and / or a TCR-α polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF0007827645000013.tif4128 a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to A nucleic acid encoding [This invention 1051] 1050. The nucleic acid of the present invention, encoding a TCR-a polypeptide comprising CDR1, CDR2, and CDR3 and / or a TCR-b polypeptide comprising CDR1, CDR2, and CDR3. [This invention 1052] 1051. The nucleic acid of the present invention, encoding a TCR-a comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:6 and / or a TCR-b comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:12. [This invention 1053] The nucleic acid of the present invention 1051 or 1052, which encodes a TCR-a comprising a CDR2 having at least 80% sequence identity to SEQ ID NO:7 and a TCR-b comprising a CDR2 having at least 80% sequence identity to SEQ ID NO:13. [This invention 1054] Any of the nucleic acids 1050 to 1053 of the present invention, encoding a TCR-a variable region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4 and / or a TCR-b variable region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10. [This invention 1055] 1054. The nucleic acid of the present invention, encoding a TCR-a polypeptide chain comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3 and / or a TCR-b chain comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:9. [This invention 1056] The nucleic acid of the present invention 1055, comprising SEQ ID NO:1 and / or SEQ ID NO:2. [This invention 1057] 1056. The nucleic acid of any one of claims 1050 to 1056, comprising a TCR-a (TRA) and a TCR-b (TRB) gene. [This invention 1058] The nucleic acid of any one of 1050 to 1057 of the present invention, which is polycistronic. [This invention 1059] 1058. The nucleic acid of the invention, comprising an internal ribosome entry site (IRES) or a P2A linker. [The present invention 1060] A nucleic acid according to any one of claims 1050 to 1059, comprising a cDNA encoding the TCR-a and / or TCR-b gene. [The present invention 1061] The nucleic acid of any one of 1050 to 1060, further encoding a polypeptide comprising a CD3 binding region. [The present invention 1062] 1061. The nucleic acid of the invention, wherein the CD3 binding region comprises a CD3-specific fragment antigen binding (Fab), a single-chain variable fragment (scFv), a single-domain antibody, or a single-chain antibody. [The present invention 1063] A nucleic acid vector comprising any one of the nucleic acids 1027 and 1050 to 1062 of the present invention. [The present invention 1064] The vector of the present invention 1063, comprising a promoter that directs expression of the nucleic acid. [This invention 1065] 1064. The vector of the present invention, wherein the promoter comprises a murine stem cell virus (MSCV) promoter. [The present invention 1066] The vector of any one of 1063 to 1065, comprising TCR-a and TCR-b genes. [This invention 1067] A cell comprising any one of the polypeptides of the present inventions 1001 to 1026, any one of the TCRs of the present inventions 1028 to 1045, 1048, or 1049, any one of the fusion proteins of the present inventions 1046 to 1049, any one of the nucleic acids of the present inventions 1027 or 1050 to 1062, or any one of the vectors of the present inventions 1063 to 1066. [The present invention 1068] The cell of the present invention 1067, including a stem cell, progenitor cell, immune cell, or natural killer (NK) cell. [The present invention 1069] The cell of the present invention 1068, including a hematopoietic stem cell or hematopoietic progenitor cell, a T cell, a cell differentiated from a mesenchymal stem cell (MSC), or an induced pluripotent stem cell (iPSC). [The present invention 1070] The cells of the invention 1068 or 1069 isolated from or derived from peripheral blood mononuclear cells (PBMC). [This invention 1071] T cells are cytotoxic T lymphocytes (CTLs), CD8 + T cells, CD4 + The cell of the invention 1069 or 1070, comprising a T cell, an invariant NK T (iNKT) cell, a gamma-delta T cell, an NKT cell, or a regulatory T cell. [This invention 1072] The cell of any one of 1067 to 1071 of the present invention, isolated from a cancer patient. [This invention 1073] A composition comprising any one of the polypeptides of the present inventions 1001 to 1026, any one of the TCRs of the present inventions 1028 to 1045, 1048, or 1049, any one of the fusion proteins of the present inventions 1046 to 1049, any one of the nucleic acids of the present inventions 1027 or 1050 to 1062, any one of the vectors of the present inventions 1063 to 1066, or any one of the cells of the present inventions 1067 to 1072. [This invention 1074] 1073. The composition of the present invention, wherein the composition is determined to be serum-free, mycoplasma-free, endotoxin-free, and / or sterile. [This invention 1075] A method for producing an engineered cell, comprising the step of transfecting a cell with any one of the nucleic acids of the present invention 1027 or 1050 to 1062 or any one of the vectors of the present invention 1063 to 1066. [This invention 1076] The method of claim 1075, further comprising culturing the cells in a medium, incubating the cells under conditions that allow the cells to divide, screening the cells, and / or freezing the cells. [This invention 1077] A method for treating cancer in a subject, comprising administering to a subject in need thereof the composition of invention 1073 or 1074 or the cell of any of inventions 1067 to 1072. [This invention 1078] The method of claim 1077, wherein the subject has previously undergone treatment for cancer. [This invention 1079] The method of claim 1078, wherein the subject has been determined to be refractory to a previous treatment. [The present invention 1080] The method of any of claims 1077 to 1079, further comprising administering an additional therapeutic agent. [This invention 1081] The method of any of claims 1077 to 1080, wherein the cancer comprises stage I, II, III, or IV cancer. [This invention 1082] 1082. The method of any one of claims 1077 to 1081, wherein the cancer comprises metastatic and / or recurrent cancer. [This invention 1083] 1083. The method of any of claims 1077 to 1082, wherein the cancer comprises glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, glioblastoma, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, or melanoma cancer. [This invention 1084] The method of claim 1083, wherein the cancer comprises glioblastoma. [This invention 1085] The method of any one of claims 1077 to 1084, wherein the subject is HLA-A2 positive and / or has been determined to be HLA-A2 positive. [The present invention 1086] The method of claim 1085, wherein the subject is HLA-A0201 positive and / or has been determined to be HLA-A0201 positive. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments and aspects of the present invention, are given by way of example only, as various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0035] [Figure 1]Generation of COL6A3-642 peptide-specific CTLs. Mature dendritic cells (mDCs) derived from PBMCs of HLA-A0201+ healthy donors were pulsed with COL6A3-642 peptide and then cocultured with autologous T cells. After two rounds of stimulation, a CD8+ / tetramer+ cell population emerged and was selected for further expansion. After expansion using the rapid expansion protocol (REP), highly purified CD8+ / tetramer CTL cells were observed. Three COL6A3-642-specific CTL cell lines, C4, E3, and G4, were generated. [Figure 2] Epitope recognition affinity assay of COL6A3-642-specific CTL cell lines. T2 cells pulsed with various concentrations of COL6A3-642 peptide were used as targets. The lytic ability of different COL6A3-642-specific CTL cell lines was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio was 20:1. [Figure 3A] Figure 3A-C: Functional validation of COL6A3-642-specific CTL cell lines. Different tumor targets, U87MG, T98G, LN-18, and M059K, were cocultured with different COL6A3-642-specific CTL cell lines. The lytic ability of COL6A3-642 CTL cell lines was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio ranged from 40:1 to 1.25:1. Both the C4 and G4 CTL cell lines were able to effectively lyse the positive targets, T98G and LN-18, but were unable to lyse the negative target, M095K. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4]Generation of COL6A3-642-specific TCR-T. Full-length TCR alpha and beta chains from the COL6A3-642 C4 CTL cell line were assembled with a P2A skip peptide and inserted into the retroviral vector pMSGV3. This recombinant retroviral vector was used to infect PBMCs from an HLA-A0201+ healthy donor. After infection, a CD8+ / tetramer+ population emerged compared with uninfected PBMCs. After tetramer-induced selection and expansion, highly purified TCR-T were generated. [Figure 5] Figure 5A-E: Functional validation of COL6A3-642-specific TCR-T cell lines. (A-D) Different tumor targets were co-cultured with COL6A3-642-specific TCR-T cell lines. The lytic ability of COL6A3-642 TCR-T cell lines was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratios ranged from 40:1 to 1.25:1. COL6A3-642 TCR-T cell lines were able to effectively lyse the positive targets T98G, LN-18, and U87MG, but were unable to lyse the negative target M095K. (E) T2 cells pulsed with various concentrations of COL6A3-642 peptide were used as targets and co-cultured with COL6A3-642 TCR-T cells. The effector-to-target (E:T) ratio was 20:1. The COL6A3-642 TCR-T cell line was able to effectively lyse targets even at very low peptide concentrations (10 pg / ml). [Figure 6] Functional detection of COL6A3-642 TCR-T by intracellular cytokine staining (ICS) assay. The COL6A3-642 TCR-T cell line was cocultured with tumor targets U87MG, LN-18, T98G, and M059K at an E:T ratio of 10:1. After overnight coculture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ, and TNF-α, were detected by ICS assay. CD137, CD69, IFN-γ, and TNF-α levels of COL6A3-642 TCR-T were significantly enhanced when the cell line was cocultured with positive targets, especially T98G, compared with negative targets. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description of the Invention The present disclosure provides a T cell receptor (TCR) that recognizes an HLA-A2-restricted epitope from the tumor antigen COL6A3, which has the amino acid sequence FLLDGSANV (SEQ ID NO:15). The present disclosure also provides a nucleotide sequence encoding the TCR, and an expression vector containing the nucleotide sequence that can be used to modify cells, such as peripheral blood mononuclear cells, to generate COL6A3-specific T cells. The present disclosure also provides the use of COL6A3-specific T cells for adoptive immunotherapy of HLA-A2-positive cancer patients whose malignant cells express the COL6A3 antigen.

[0037] I. Engineered T Cell Receptors T cell receptors contain two distinct polypeptide chains, termed the T cell receptor α (TCRα) and β (TCRβ), linked by disulfide bonds. These α:β heterodimers are structurally very similar to the Fab fragments of immunoglobulin molecules and are responsible for antigen recognition by most T cells. A small number of T cells have a separate, but structurally similar, receptor composed of a pair of distinct polypeptide chains, termed γ and δ. Both types of T cell receptors differ from membrane-bound immunoglobulins that function as B cell receptors: T cell receptors have only one antigen-binding site, whereas B cell receptors have two, and T cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.

[0038] Both chains of the T cell receptor have an amino-terminal variable (V) region with homology to immunoglobulin V domains, a constant (C) region with homology to immunoglobulin C domains, and a short hinge region containing cysteine ​​residues that form interchain disulfide bonds. Each chain spans the lipid bilayer by a hydrophobic transmembrane domain and terminates in a short cytoplasmic tail.

[0039] The three-dimensional structure of the T cell receptor has been determined. This structure is certainly similar to that of an antibody Fab fragment, as inferred from previous studies of the gene encoding the T cell receptor. The chains of the T cell receptor fold in much the same way as those of the Fab fragment, although the final structure appears to be slightly shorter and wider. However, there are several distinct differences between the T cell receptor and the Fab fragment. The most notable difference is in the Cα domain, which folds differently from any other immunoglobulin-like domain. Half of the domain juxtaposed with the Cβ domain forms a β-sheet similar to that found in other immunoglobulin-like domains, while the other half of the domain is formed by loosely packed chains and a short segment of α-helix. While an intramolecular disulfide bond typically connects two β-strands in immunoglobulin-like domains, in the Cα domain, it connects the β-strand to this α-helical segment.

[0040] There are also differences in the way the domains interact. The interface between the V and C domains of both T cell receptor chains is more extensive than in antibodies, potentially reducing the flexibility of the hinge junction between the domains. The interaction between the Cα and Cβ domains is unique in that it is carbohydrate-assisted, with sugar groups from the Cα domain making several hydrogen bonds to the Cβ domain. Finally, comparison of the variable binding sites shows that the complementarity-determining region (CDR) loops align fairly closely with those of antibody molecules, but there is some displacement compared to the loops of antibody molecules. This displacement is particularly pronounced in the Vα CDR2 loop, which is oriented nearly perpendicular to the equivalent loop in the antibody V domain as a result of a shift in the β strand that anchors one end of the loop from one face of the domain to the other. Strand displacement also causes changes in the orientation of the Vβ CDR2 loop in two of the seven Vβ domains with known structures. To date, only seven T cell receptor crystal structures have been solved at this level of resolution.

[0041] Aspects of the present disclosure relate to engineered T cell receptors. The term "engineered" refers to T cell receptors having a TCR variable region grafted onto a TCR constant region to create chimeric polypeptides that bind to the peptides and antigens of the present disclosure. In certain aspects, the TCR is used for cloning, enhancing expression, detection, or therapeutic control of the construct, but includes intervening sequences not present in the endogenous TCR, such as a multiple cloning site, a linker, a hinge sequence, a modified hinge sequence, a modified transmembrane sequence, a detection polypeptide or molecule, or a therapeutic control that may allow for the selection or screening of cells containing the TCR.

[0042] In some aspects, the TCR comprises non-TCR sequences. Thus, certain aspects relate to TCRs having sequences not derived from TCR genes. In some aspects, the TCR is chimeric in that it comprises sequences normally found in TCR genes, but comprises sequences from at least two TCR genes that are not necessarily found together in nature.

[0043] In some aspects, the engineered TCRs of the present disclosure comprise the variables shown below: TIFF0007827645000014.tif242159TIFF0007827645000015.tif242159TIFF0007827645000016.tif198159

[0044] The following table characterizes the TCR-a aspects: TIFF0007827645000017.tif52160

[0045] The following table characterizes aspects of TCR-b: TIFF0007827645000018.tif59160

[0046] II. Proteinaceous Compositions As used herein, "protein," "peptide," or "polypeptide" refers to a molecule containing 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, a wild-type version of a protein or polypeptide is utilized; however, in many aspects of the present disclosure, a modified protein or polypeptide is utilized to generate an immune response. The above terms may be used interchangeably. A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative 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 a protein or polypeptide can 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 but retain wild-type activity or function in other respects, such as immunogenicity.

[0047] When a protein is specifically referred to herein, it generally refers to a native (wild-type) or recombinant (modified) protein, or a protein, optionally with any signal sequence removed. Proteins can be isolated directly from the organism in which they occur, 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 encoding polypeptides (e.g., antibodies or fragments thereof). While the term "recombinant" can be used in conjunction with the name of a polypeptide or a specific polypeptide, it generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule.

[0048] In certain aspects, the size of the protein or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid or nucleic acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that polypeptides may be mutated by truncation to be shorter than their corresponding wild-type form, and may be modified by fusing or conjugating heterologous proteins or polypeptide sequences with specific functions (e.g., for targeting or localization, to enhance immunogenicity, for purification purposes, etc.).

[0049] A polypeptide, protein of the disclosure, or a polynucleotide encoding such a polypeptide or protein, may contain 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 range derivable therein) or more variant amino acid or nucleic acid substitutions, or may contain any of the variant amino acid or nucleic acid substitutions set forth in SEQ ID NO: No.: 1 to 17, at least 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 consecutive amino acids or nucleic acids, or any range derivable therein, 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, 9 3, 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, 1 26, 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, 7, 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 consecutive amino acids or nucleic acids, or derivable therein The peptide or polypeptide may 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% similar, identical, or homologous to any range derivable therein. In certain aspects, the peptide or polypeptide is a human sequence or is based on a human sequence. In certain aspects, the peptide or polypeptide is non-naturally occurring and / or in a combination of peptides or polypeptides.

[0050] In some aspects, the protein, polypeptide, or nucleic acid is selected from the group consisting of SEQ ID NO: NO: 1-17, 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, 1 1, 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 or 320 amino acids or nucleotides (or any range derivable therein).

[0051] In some aspects, the protein, polypeptide, or nucleic acid is selected from the group consisting of SEQ ID NO: NO: 1-17, 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, 1 1, 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 or 320 amino acids or nucleotides (or any range derivable therein), and 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% for one of NO: 1-17 (or any range derivable therein), 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 range derivable therein).

[0052] In some aspects, the protein, polypeptide, or nucleic acid is selected from the group consisting of SEQ ID NO: NO: 1 to 17, 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, or 320 (or any range derivable therein), at least 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 6, 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, 1 22, 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, 3, 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, 2 78, 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, or 320 (or any range derivable therein), or 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, 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, 1109, 1110, 1120, 1121, 1122, 1123, 1124, 1125, 11 2, 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 6, 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, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 31 48, 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 or 320 (or any range derivable therein) consecutive amino acids or nucleic acids.

[0053] In some aspects, a polypeptide, protein, or nucleic acid 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% similarity to one of SEQ ID NOs: 1-17. (or any range derivable therein), at most 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 range derivable therein), or is 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 range derivable therein) similar, identical or homologous to a SEQ ID NO: 1 to 17, at least 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, 43, 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 4, 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, 68, 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, 29 9, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any range derivable therein), 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、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 or 320 (or any range derivable therein), 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, 67, 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 8, 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 It may comprise 5, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319 or 320 (or any range derivable therein) consecutive amino acids.

[0054] In some aspects, the SEQ ID NO: 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 1, 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 6, 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, 1 47, 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, 20 8, 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, 2 39, 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, starting at position 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 or 950 and SEQ ID No.: At least 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 of any of Nos. 1 to 17 , 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, 8 70, 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, 90 1, 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, or 950 (or any range derivable therein), at most 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, 9 6, 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 8, 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 or 950 (or any range derivable therein), 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, 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, 9 5, 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, 1 90, 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 1, 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, 06, 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, There is a nucleic acid molecule or polypeptide that comprises 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, or 950 (or any range derivable therein) consecutive amino acids or nucleotides.

[0055] Nucleotide and protein, polypeptide, and peptide sequences of various genes have been previously disclosed and can be found in recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information's Genbank and GenPept databases (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as would be known to one skilled in the art.

[0056] It is contemplated that the compositions of the present disclosure will have from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition may be 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), at least 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) 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).

[0057] The following is a discussion of altering the amino acid subunits of proteins to create equivalent or, in some cases, improved second-generation variant polypeptides or peptides. For example, certain amino acids can be substituted for other amino acids in a protein or polypeptide sequence, with or without significant loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. Because the functional activity of a protein is determined by the interaction ability and properties of the protein, certain amino acid substitutions can be made in the protein sequence and its corresponding DNA coding sequence, and still produce a protein with similar or desirable properties. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene encoding a protein without significantly losing its biological usefulness or activity.

[0058] 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. "Neutral substitutions" or "neutral mutations," which refer to changes in a codon that encode a biologically equivalent amino acid, are also contemplated.

[0059] The amino acid sequence variants of the disclosure can be substitutional, insertional, or deletional variants. Mutations in the polypeptides of the disclosure can 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 (or any range derivable therein) non-contiguous or contiguous amino acids of the protein or polypeptide compared to the wild-type. Variants can include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical to any sequence provided or referenced herein, including all values ​​and ranges therebetween. Variants can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substituted amino acids.

[0060] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially identical as set forth in one of the sequences disclosed herein, so long as they meet the above criteria, including maintenance of the biological protein activity involved in expression of the protein. The addition of terminal sequences applies particularly to nucleic acid sequences, which can include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0061] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or several adjacent amino acids may be deleted. A stop codon may be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate a truncated protein.

[0062] Insertional variants typically involve the addition of an amino acid residue at a non-terminal point in the polypeptide. This can include the insertion of one or more amino acid residues. Terminal additions can also be made, and these can include fusion proteins, which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0063] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide and may be designed to modify one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, i.e., one amino acid may be substituted with an amino acid of similar chemical properties. A "conservative amino acid substitution" may involve the exchange of a member of one amino acid class for another member of the same class. Conservative substitutions are well known in the art, and include, for example, alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, 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 or methionine, 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 include non-naturally occurring amino acid residues, which are usually incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted amino acid moieties.

[0064] Alternatively, substitutions may be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing one amino acid residue with a chemically different residue, such as substituting a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member from another class.

[0065] Those skilled in the art can use well-known techniques to determine suitable variants of polypeptides as described herein.Those skilled in the art can identify suitable regions of molecules that can be changed without destroying activity by targeting regions that are not considered important for activity.Those skilled in the art can also identify amino acid residues and molecular parts that are conserved between similar proteins or polypeptides.In a further aspect, regions that may be important for biological activity or structure can undergo conservative amino acid substitutions without significantly changing biological activity or adversely affecting protein or polypeptide structure.

[0066] When making such changes, the hydropathic index of the amino acids may be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then iteratively averaging these values ​​along the peptide chain. Each amino acid is 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function to 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 amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids can be substituted for other amino acids having similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on hydropathic index, certain aspects include substitution of amino acids whose hydropathic index is within ±2. In some aspects of the invention, within ±1 is included, and in other aspects of the invention, within ±0.5 is included.

[0067] It is also understood in the art that similar amino acids can be effectively substituted based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigen binding, i.e., as the biological properties of the protein. These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+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). Making changes based on similar hydrophilicity values ​​includes, in certain aspects, substitution of amino acids with hydrophilicity values ​​within ±2, in other aspects within ±1, and in still other aspects within ±0.5. In some cases, epitopes can be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still result in a biologically equivalent and immunologically equivalent protein.

[0068] Moreover, one skilled in the art can review structure-function studies to identify residues in similar polypeptides or proteins that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for the activity or structure of the similar protein. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0069] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to the structure of similar proteins or polypeptides. Given such information, one skilled in the art can 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 alter amino acid residues predicted to be on the surface of the protein, as such residues may be involved in important interactions with other molecules. Furthermore, one skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue position. These variants can then be screened using standard assays for binding and / or activity, thus obtaining information gleaned from such routine experiments that allows one skilled in the art to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0070] In some aspects of the present invention, amino acid substitutions are made to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in portions of antibodies outside of the domains that form intermolecular contacts. In such aspects, conservative amino acid substitutions that do not substantially change the structural characteristics of proteins or polypeptides (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes natural antibodies) can be used.

[0071] III. Nucleic acids In certain aspects, nucleic acid sequences can exist in a variety of instances, including: isolated segments of integrated sequences or recombinant polynucleotides and recombinant vectors encoding one or both chains of an antibody, or fragments, derivatives, muteins, or variants thereof; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting expression of polynucleotides; and complementary sequences of the foregoing described herein. Nucleic acids encoding epitopes for certain antibodies provided herein are also provided. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0072] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from total genomic nucleic acid. Included within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in some aspects, contains regulatory sequences that are substantially isolated from a naturally occurring gene or protein-coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present in a polynucleotide.

[0073] In this regard, the terms "gene," "polynucleotide," or "nucleic acid" are used to refer to a nucleic acid (including any sequences required for proper transcription, post-translational modification, or localization) that encodes a protein, polypeptide, or peptide. As will be understood by those of skill in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or can be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide can include a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide can be encoded by a nucleic acid, including variants, that have slightly different nucleic acid sequences but still encode the same or substantially similar protein.

[0074] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein, wherein the variants comprise at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values ​​and ranges between, compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis with standard parameters). In one aspect, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95%, and more identity over the entire length of the sequence to the amino acid sequences described herein; or a nucleotide sequence complementary to the isolated polynucleotide.

[0075] Regardless of the length of the coding sequence itself, nucleic acid segments may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and therefore their total length may vary considerably. Nucleic acids can be of 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 contain one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Thus, it is contemplated that nucleic acid fragments of almost any length can be used, with the total length preferably being limited by the ease of purification and the intended use in the recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification, transport, secretion, post-translational modification of the polypeptide, or to allow for therapeutic utility such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0076] A. Hybridization A nucleic acid that hybridizes to another nucleic acid under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. For example, see Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those that use a hybridization temperature of 42°C and contain about 50% formamide), and a washing condition of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions involve hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one skilled in the art can manipulate hybridization and / or wash conditions to increase or decrease the stringency of hybridization such that nucleic acids containing nucleotide sequences 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 will typically remain hybridized to each other.

[0077] Parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are described, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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 incorporated by reference in their entireties for all purposes), and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0078] B. Mutation Changes can be introduced into nucleic acids by mutation, thereby resulting in changes in the amino acid sequence of the polypeptide (e.g., antibody or antibody derivative) encoded by the nucleic acid. Mutations can be introduced using any technique known in the art. In one aspect, one or more specific amino acid residues are changed, for example, using a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. Regardless of how they are generated, mutant polypeptides can be expressed and screened for desired properties.

[0079] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the polypeptides encoded by the nucleic acids. For example, nucleotide substitutions resulting in amino acid substitutions at non-essential amino acid residues can be made. Alternatively, one or more mutations can be introduced into nucleic acids that selectively alter the biological activity of the encoded polypeptides. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, reducing, or eliminating activity. Examples of qualitative changes include altering the antigen specificity of antibodies.

[0080] C. Probe In another aspect, the nucleic acid molecule is suitable for use as a primer or hybridization probe for the detection of a nucleic acid sequence. The nucleic acid molecule can comprise a portion of a nucleic acid sequence encoding a full-length polypeptide, e.g., a fragment that can be used as a probe or primer, or only a fragment that encodes an active portion of a given polypeptide.

[0081] In another aspect, the nucleic acid molecule can be used as a probe or PCR primer for a specific antibody sequence. For example, a nucleic acid molecule probe can be used in a diagnostic method, or a nucleic acid molecule PCR primer can be used to amplify a region of DNA that can be used to isolate a nucleic acid sequence for use in producing an antibody variable domain, among others. See, for example, Gaily Kivi et al., BMC Biotechnol. 16:2 (2016). In a preferred aspect, the nucleic acid molecule is an oligonucleotide. In a more preferred aspect, the oligonucleotide is derived from the highly variable regions of the heavy and light chains or alpha and beta chains of the antibody or TCR of interest. In an even more preferred aspect, the oligonucleotide encodes all or part of one or more CDRs or TCRs.

[0082] A probe based on the desired sequence of the nucleic acid can be used to detect the nucleic acid or a similar nucleic acid, for example, a transcript encoding a polypeptide of interest. The probe can contain a label group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such a probe can be used to identify cells expressing the polypeptide.

[0083] IV. Polypeptide Expression In some aspects, nucleic acid molecules (e.g., TCR genes) encoding the polypeptides or peptides of the present disclosure can be made by methods known in the art, such as by isolating from B cells of immunized and isolated mice, expressing them as phage displays in any suitable recombinant expression system, and allowing them to assemble to form antibody molecules, or by recombinant methods.

[0084] A. Expression The nucleic acid molecule can be used to express large amounts of the polypeptide. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule can be used for humanization of the TCR gene.

[0085] B. Vector In some aspects, expression vectors containing nucleic acid molecules encoding a polypeptide of a desired sequence or a portion thereof (e.g., a fragment comprising one or more CDRs or one or more variable region domains) are contemplated. Expression vectors containing nucleic acid molecules may encode a heavy chain, a light chain, an alpha chain, a beta chain, or an antigen-binding portion thereof. In some aspects, expression vectors containing nucleic acid molecules may encode fusion proteins, modified antibodies, 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.

[0086] To express a polypeptide or peptide of the present disclosure, DNA encoding the polypeptide or peptide is inserted into an expression vector so that the gene region is operably linked to transcriptional and translational control sequences. In some aspects, the vector encodes a functionally complete human CH or CL immunoglobulin or TCR sequence with appropriate restriction sites engineered to allow for easy insertion and expression of any variable region sequence. In some aspects, the vector encodes a functionally complete human TCR alpha or TCR beta sequence with appropriate restriction sites engineered to allow for easy insertion and expression of any variable sequence or CDR1, CDR2, and / or CDR3. Typically, expression vectors used in any host cell contain sequences for plasmid or viral maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences," typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Such sequences and methods for their use are well known in the art.

[0087] C. Expression Systems Numerous expression systems exist that contain at least some or all of the above-mentioned expression vectors. Prokaryotic and / or eukaryotic systems can be used in one aspect to produce nucleic acid sequences or their cognate polypeptides, proteins, and peptides. Commercially and widely available systems include, but are not limited to, bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. Those skilled in the art can express the vector using an appropriate expression system to produce the nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0088] V. Methods of Gene Transfer Suitable methods for nucleic acid delivery to result in expression of the composition are expected to include virtually any method by which nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into a cell, tissue, or organism, as described herein or known to those of skill in the art. Such methods include microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference), by injection (U.S. Patent Nos. 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 of which is 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; Nicolau et 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; Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al., each of which is incorporated herein by reference);Direct delivery of DNA, such as by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); or by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0089] A. host cell Another aspect contemplates the use of host cells into which recombinant expression vectors have been introduced. Antibodies can be expressed in a variety of cell types. Antibody-encoding expression constructs can be transfected into cells by various methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may utilize control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain aspects, antibody expression constructs can be placed under the control of promoters associated with T cell activation, such as those controlled by NFAT-1 or NF-κB, transcription factors that can both be activated upon T cell activation. Control of antibody expression allows T cells, such as tumor-targeting T cells, to sense their surroundings and to effect real-time regulation of cytokine signaling in both the T cell itself and surrounding endogenous immune cells. Those skilled in the art will understand the conditions for incubating and maintaining host cells to allow vector replication. Techniques and conditions that allow for large-scale production of vectors and production of the nucleic acid encoded by the vector and its cognate polypeptide, protein, or peptide are also understood and known.

[0090] It is known that for stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of cells can integrate foreign DNA into their genome.In order to identify and select these integrants, selectable markers (for example, for antibiotic resistance) are generally introduced into host cells together with the gene of interest.The cells that have stably transfected introduced nucleic acid can be identified by drug selection, among other methods known in the art (for example, the cells that have integrated selectable marker gene survive, while other cells die).

[0091] B. Isolation Nucleic acid molecules encoding the entire heavy, light, alpha, and beta chains of an antibody or TCR, or one or both of their variable regions, can be obtained from any source that produces antibodies. Methods for isolating mRNA encoding antibodies are well known in the art. See, for example, Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, for example, Kabat et al., 1991, supra. The nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed in cells into which they have been introduced, and the antibody can be isolated.

[0092] VI. Further Treatments A. Immunotherapy In some aspects, the method includes the administration of an additional therapy. In some aspects, the additional therapy includes cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be classified as active, passive, or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface known as tumor-associated antigens (TAAs) that can be detected by the immune system; these are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapies are known in the art, and some are described below.

[0093] 1. Checkpoint inhibitors and combination treatments Aspects of the disclosure can include administration of immune checkpoint inhibitors, which are further described below.

[0094] a. PD-1, PDL1, and PDL2 inhibitors PD-1 can act in the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 in epithelial cells and tumor cells. PDL2 is expressed in macrophages and dendritic cells. The primary role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to tissues during immune responses. The inhibitors disclosed herein can block one or more functions of PD-1 and / or PDL1 activity.

[0095] 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.

[0096] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain aspects, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another aspect, the PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its ligand-binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its ligand-binding partner. In certain aspects, the PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, as described in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0097] 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 the extracellular portion 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 is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0098] 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 a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0099] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Thus, 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 to the same epitope on PD-1, PDL1, or PDL2 as the aforementioned antibodies and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the aforementioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) variable region amino acid sequence identity to the aforementioned antibodies.

[0100] b. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds 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 inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors of the present disclosure can block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitors block the interaction between CTLA-4 and B7-1. In some aspects, the inhibitors block the interaction between CTLA-4 and B7-2.

[0101] 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 an oligopeptide.

[0102] Anti-human CTLA-4 antibodies (or VH domains and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be produced 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 U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the methods disclosed herein. The disclosures of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0103] An additional 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., WO01 / 14424).

[0104] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, 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 to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) variable region amino acid sequence identity to the aforementioned antibodies.

[0105] 2. Inhibition of costimulatory molecules In some aspects, the immunotherapy includes inhibitors of costimulatory molecules. In some aspects, the inhibitors include inhibitors of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0106] 3. Dendritic Cell Therapy Dendritic cell therapy induces an anti-tumor response by having dendritic cells present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating them to kill other cells that present the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help target cancer antigens. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0107] One way to induce dendritic cells to present tumor antigens is by vaccinating with autologous tumor lysates or short peptides (small portions of proteins that correspond to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to enhance immune and antitumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0108] Dendritic cells can also be activated in vivo by expressing GM-CSF in tumor cells, which can be achieved by genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0109] Another strategy is to remove dendritic cells from the patient's blood and activate them ex vivo. The dendritic cells are activated in the presence of tumor antigens, which can be single tumor-specific peptides / proteins or tumor cell lysates (a solution of destroyed tumor cells). These cells (with optional adjuvants) are infused to elicit an immune response.

[0110] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, inducing dendritic cells to mature and providing immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 have been used as antibody targets.

[0111] 4. CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are engineered receptors that combine immune cells with new specificities to target cancer cells. Typically, these receptors transfer the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they combine parts from different sources. CAR-T cell therapy refers to the use of such transformed cells for cancer treatment.

[0112] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to engineer T cells that target markers found on cancer cells. Scientists can remove T cells from a person, genetically modify them, and inject them back into the patient to attack cancer cells. Once engineered, T cells act as a "living drug." CAR-T cells create a link between their extracellular ligand-recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand-recognition domain is typically a single-chain variable fragment (scFv). A key aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells, and not normal cells, are targeted. The specificity of CAR-T cells is determined by the choice of the molecule being targeted.

[0113] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta). In some aspects, the CAR-T therapy targets CD19.

[0114] 5. Cytokine therapy Cytokines are proteins produced by many types of cells present in tumors. They can modulate the immune response. Tumors often use cytokines to promote tumor growth and reduce the immune response. These immunomodulatory effects allow them to be used as drugs to elicit an immune response. Two commonly used cytokines are interferons and interleukins.

[0115] Interferons are produced by the immune system. They are usually involved in antiviral responses, but are also used in cancer treatment. They are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ), and type III (IFNλ).

[0116] Interleukins have numerous immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0117] 6. Adoptive T cell therapy Adoptive T cell therapy is a form of passive immunization through the transfusion of T cells (adoptive cell transfer). T cells are found in the blood and tissues and are typically activated when they encounter foreign pathogens. Specifically, T cells become activated when their surface receptors encounter cells that display a portion of a foreign protein on their surface antigens. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal and tumor tissues, in which case 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 tumors, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0118] Several methods have been developed to generate and obtain tumor-targeting T cells. T cells specific for tumor antigens can be depleted from tumor samples (TIL) or filtered from the blood. Subsequent activation and culture are performed ex vivo, followed by reinfusion. Activation can be achieved through gene therapy or by exposing T cells to tumor antigens.

[0119] B. Chemotherapy In some aspects, the additional therapy comprises chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (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 substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine), and purine analogs and related substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); Natural products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), and (d) various agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0120] Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer, or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as intravenous, subcutaneous, intratumoral, or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain aspects, effective doses used in clinical applications include about 15 mg / m to about 20 mg / m for 5 days every 3 weeks for a total of 3 courses. In some aspects, the amount of cisplatin delivered to cells and / or subjects in conjunction with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount that would be delivered if cisplatin were used alone.

[0121] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector and doxorubicin has been shown to be effective in overcoming resistance to chemotherapy and / or TNFα, suggesting that combined treatment with the construct and doxorubicin will overcome resistance to both doxorubicin and TNFα.

[0122] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain aspects, suitable intravenous doses for adults include about 60 mg / m² to about 75 mg / m² at intervals of about 21 days, or about 25 mg / m² to about 30 mg / m² on each of two or three consecutive days repeated at intervals of about 3 to about 4 weeks, or about 20 mg / m² once a week. The lowest doses should be used in elderly patients if there is previous myelosuppression or neoplastic bone marrow infiltration caused by previous chemotherapy, or if the drug is combined with other myelopoiesis-suppressing drugs.

[0123] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards can include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN® available from Mead Johnson, NEOSTAR® available from Adria) is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, about 40 mg / kg to about 50 mg / kg initially in divided doses over about 2 to about 5 days, or about 10 mg / kg to about 15 mg / kg every about 7 to about 10 days, or about 3 mg / kg to about 5 mg / kg twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal effects, the intravenous route is preferred. Drugs may also be administered intramuscularly, by infiltration, or into body cavities.

[0124] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU can be administered to a subject at a dosage of anywhere from about 7.5 to about 1000 mg / m. Furthermore, 5-FU dosing schedules can be for various periods, for example, up to 6 weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0125] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and therefore may be useful in the present disclosure for these cancers as well.

[0126] The amount of chemotherapeutic agent delivered to a patient can vary. In one suitable aspect, the chemotherapeutic agent can be administered in an amount effective to cause the arrest or regression of cancer in a host when the chemotherapy is administered in conjunction with the construct. In other aspects, the chemotherapeutic agent can be administered in an amount anywhere from 2 to 10,000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. For example, the chemotherapeutic agent can be administered in an amount about 20 times less, about 500 times less, or even about 5000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct and to determine effective dosages. For example, such compounds can be tested in appropriate animal model systems, including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, etc., prior to human testing. As described in the Examples, in vitro testing can also be used to determine appropriate combinations and dosages.

[0127] C. Radiation therapy In some aspects, the additional or previous treatment comprises radiation, such as ionizing radiation. As used herein, "ionizing radiation" refers to radiation that contains particles or photons that have sufficient energy or can generate sufficient energy through nuclear interaction to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is x-rays. Means for delivering x-rays to target tissues or cells are well known in the art.

[0128] D. Surgery In some aspects, the additional treatment includes surgery. Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as treatments of aspects of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0129] Removal of part or all of cancer cells, tissues, or tumors can result in the formation of a cavity in the body. Treatment can be performed by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or any range derivable therein). These treatments can also be at various dosages.

[0130] VII. DETECTION AND THERAPEUTIC AGENTS In some aspects of the present disclosure, it may be useful to detectably or therapeutically label the TCR or fusion protein of the present disclosure.Methods for conjugating polypeptides to these agents are known in the art.For illustrative purposes only, polypeptides can be labeled with detectable moieties such as radioactive atoms, chromophores, fluorophores, etc.Such labeled polypeptides can be used in diagnostic techniques in vivo, or in isolated test samples, or in the methods described herein.

[0131] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to a composition to be detected, e.g., a protein such as a polynucleotide or antibody, to produce a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP). The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Suitable labels therefore include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected or quantified. A simply detected response generally includes a response that merely confirms its presence, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can occur directly, using a luminophore or fluorophore associated with the assay component actually responsible for binding, or indirectly, using a luminophore or fluorophore associated with another component (e.g., a reporter or indicator).

[0132] Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally involves a change in, or the generation of, a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th(ed.) Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0133] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th (ed.).

[0134] In another aspect, the fluorescent label is functionalized to facilitate covalent binding to cellular components present in or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label will depend on the site of attachment to either the linker, agent, marker, or second labeling agent.

[0135] The attachment of the fluorescent label can be direct to the cellular component or compound, or alternatively via a linker. Suitable binding pairs for indirectly linking the fluorescent label to an intermediate include, but are not limited to, antigen / polypeptide, such as rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0136] Coupling of the polypeptide to a low molecular weight hapten can increase the sensitivity of the antibody in an assay. The hapten can then be specifically detected by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten polypeptides. See Harlow and Lane (1988), supra.

[0137] The conjugated agent can be linked to the polypeptide directly or indirectly using any of a number of available methods. For example, the agent can be attached to the hinge region of a reduced antibody component via disulfide bond formation using a cross-linking agent such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety in the Fc region of the antibody (Yu et al., 1994; Upeslacis et al., 1995; Price, 1995).

[0138] Techniques for conjugating agents to polypeptides are well known (Amon et al., 1985; Hellstrom et al., 1987; Thorpe, 1985; Baldwin et al., 1985; Thorpe et al., 1982).

[0139] The polypeptides of the present disclosure or antigen-binding regions thereof can be linked to another functional molecule such as a ligand, a cytotoxic molecule, a chemotherapeutic agent, or other agent described as an additional therapeutic substance.

[0140] VIII. Cell Formulation and Culture In certain aspects, the cells of the present disclosure may be specially formulated and / or they may be cultured in a particular medium. The cells may be formulated in a manner that makes them suitable for delivery to a recipient without adverse effects.

[0141] In certain aspects, the medium may be prepared using a medium used to culture animal cells as the basal medium, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, 199 Medium, Eagle's MEM, αMEM, DMEM, Ham's, RPMI-1640, and Fisher's Medium, as well as any combination thereof, but the medium is not particularly limited thereto as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0142] The medium can be serum-containing or serum-free, or xeno-free. To prevent contamination with components derived from different animals, the serum can be derived from the same animal as that of the stem cells. Serum-free medium refers to a medium that does not contain raw or unpurified serum, and therefore can include a medium that contains purified blood-derived components or animal tissue-derived components (such as growth factors).

[0143] The medium may or may not contain any serum substitute. Serum substitutes may include materials that appropriately contain albumin (such as albumin substitutes, such as lipid-rich albumin, bovine albumin, recombinant albumin, or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes may be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, for greater convenience, any commercially available material may be used. Commercially available materials include Knockout Serum Replacement (KSR), Chemically-Defined Lipid Concentrated (Gibco), and Glutamax (Gibco).

[0144] In certain aspects, the medium may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of the following: biotin; DL-alpha tocopherol acetate; DL-alpha-tocopherol; vitamins such as vitamin A (acetate); BSA (bovine serum albumin) or human albumin, fatty acid-free fraction V; catalase; human recombinant insulin; human transferrin; proteins such as superoxide dismutase; corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or other components such as T3 (triiodo-I-thyronine). In certain aspects, one or more of these may be explicitly excluded.

[0145] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any range derivable therein): biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12, or the medium comprises a combination thereof or a salt thereof. In some aspects, the medium comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamin comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or a combination or salt thereof. In some aspects, the medium further comprises a protein. In some aspects, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some aspects, the medium further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof. In some aspects, the medium comprises one or more of B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or a combination thereof. In some aspects, the medium comprises or further comprises amino acids, simple sugars, inorganic ions.In some aspects, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In some aspects, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In some aspects, the medium further includes one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein, and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), simple sugars, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain aspects, one or more of these may be explicitly excluded.

[0146] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, of which, in certain aspects, one or more may be explicitly excluded.

[0147] One or more of the media components has a concentration of at least 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein, and at most 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 It may be added at a concentration of ng / L, ng / ml, μg / ml, mg / ml or any range derivable therein, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml or any range derivable therein.

[0148] In certain aspects, the cells of the present disclosure are specially formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single-dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage before use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, and certain formulations include 2.5% human albumin. The cells may be specially formulated for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In certain aspects, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more after thawing.

[0149] In certain aspects, the cells of the present disclosure comprise an exogenous TCR, which can have a defined antigen specificity. In some aspects, the TCR can be selected based on the lack or reduced alloreactivity to the intended recipient (examples include certain virus-specific TCRs, xenospecific TCRs, or cancer-testis antigen-specific TCRs). In instances where the exogenous TCR is non-alloreactive, during T cell differentiation, the exogenous TCR suppresses the rearrangement and / or expression of the endogenous TCR locus through a developmental process called allelic exclusion, resulting in T cells that only express non-alloreactive exogenous TCRs and thus become non-alloreactive. In some aspects, the selection of the exogenous TCR may not necessarily be determined based on the lack of alloreactivity. In some aspects, the endogenous TCR gene is modified by genome editing so that it does not express the protein. Methods of gene editing, such as those using the CRISPR / Cas9 system, are known in the art and are described herein.

[0150] In some aspects, the cells of the present disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which CARs can be directed include, for example, at least 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, ErbB2 (HER2), EGFR family including EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-α, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, and glypican-3. (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV Examples of CARs include E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). CARs may be first-, second-, third-, or higher-generation CARs. CARs may be bispecific for any two non-identical antigens, or specific for three or more non-identical antigens.

[0151] IX. Administration of Therapeutic Compositions The therapeutic methods provided herein can include the administration of a combination of therapeutic agents, such as a first cancer therapeutic method and a second cancer therapeutic method.The therapeutic methods can be administered by any suitable method known in the art.For example, the first and second cancer treatments can be administered sequentially (at different times) or simultaneously (at the same time).In some aspects, the first and second cancer treatments are administered in separate compositions.In some aspects, the first and second cancer treatments are administered in the same composition.

[0152] Aspects of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapies can be administered in one composition, or in two or more compositions, such as two, three, or four compositions. Various combinations of agents can be utilized.

[0153] The therapeutic compositions of the present disclosure may be administered by the same or different routes of administration. In some aspects, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0154] Treatments can include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the skill of those in the clinical arts to determine the amount. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0155] Precise amounts of a therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dosage include the physical and clinical condition of the patient, the route of administration, the intended treatment goal (palliation of symptoms vs. cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatment the subject may be receiving.

[0156] Cancers suitable for treatment include, but are not limited to, tumors of all types, locations, sizes, and characteristics. In some aspects, the cancer comprises a solid tumor. In some aspects, the method relates to reducing tumor volume or treating recurrent and / or metastatic cancer. The methods and compositions of the present disclosure are useful for treating, for example, pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumor brain tumor, visual pathway and hypothalamic glioma, breast cancer, lymphatic cancer, and the like. Cancer, bronchial adenoma / carcinoid, tracheal cancer, lung cancer, Burkitt's lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal cancer of unknown primary, lymphoma of the central nervous system, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's disease, childhood extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanomaEye cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumors: extracranial, extragonadal or ovarian, gestational trophoblastic tumor, brainstem glioma, glioma, cerebral astrocytoma in children, visual pathway and hypothalamic glioma in children, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma in children, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphoblastic (also called acute lymphocytic) leukemia, acute myeloid (acute myelogenous) leukemia leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), chronic myelogenous (also called chronic myeloid leukemia), hairy cell carcinoma of the lip and oral cavity, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin (an older classification of all lymphomas other than Hodgkin) lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma in children, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma in adults, mesothelioma in children, metastatic cervical squamousNeck cancer, oral cavity (mouth) cancer, multiple endocrine neoplasia, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia in adults, acute myeloid leukemia in children, multiple myeloma, chronic myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (superficial epithelial and stromal tumors), ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, islet cell cancer of the paranasal sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma in children, plasma cell neoplasm / multiple myeloma, pleuropulmonary It is suitable for treating blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, pediatric salivary gland carcinoma sarcoma, Ewing's sarcoma family of tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine Sezary syndrome sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, Merkel cell small cell lung cancer, cancer of the small intestine, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the cervix of unknown primary, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, pediatric T-cell lymphoma, testicular cancer, throat cancer, thymoma, pediatric thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma of the endometrium, vaginal cancer, glioma of the visual pathway and hypothalamus, pediatric vulvar cancer, and Wilms' tumor (kidney cancer).

[0157] X. Kit Certain aspects of the present invention also relate to kits comprising the disclosed compositions or compositions for practicing the methods of the present invention. In some aspects, the kits can be used to assess one or more biomarkers or HLA types. In certain aspects, the kits 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, 50, 100, 500, 1,000 or more profiles. 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, 0, 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, or 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.

[0158] The kit may include components that may be individually packaged or disposed in containers, such as tubes, bottles, vials, syringes, or other suitable container means.

[0159] Individual components may be provided in the kit in concentrated amounts; in some aspects, components are provided individually at the same concentration as they are in solution with the other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.

[0160] In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. Additionally, the kits can include samples that are negative or positive controls for methylation of one or more biomarkers.

[0161] 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 as originally filed are intended to cover any claim that is multiple dependent on any one or combination of the claims as filed. [Example]

[0162] XI. Working Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will recognize that the procedures disclosed in the examples below are procedures found by the inventors to work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the disclosure and still obtain a like or similar result.

[0163] Example 1: Generation of TCR gene-modified T cells with the TCR sequence for adoptive T cell therapy to treat HLA-A2-positive cancer patients An HLA-A2-restricted epitope derived from the human tumor antigen COL6A3 (amino acid sequence: FLLDGSANV (SEQ ID NO:15)) has previously been identified. Herein, we describe tools that can be used to target this epitope, including engineered TCRs and cells bearing those TCRs. Using this peptide and the corresponding tetramer, we successfully generated antigen-specific CTL cell lines in vitro (Figure 1). These cell lines were able to kill tumor cells that naturally presented this peptide (Figures 2-3). Based on this finding, we cloned the TCR sequence (including the TCR alpha and beta chains) from the CTL cell line and then constructed retroviral vectors containing the full-length TCR alpha and beta chains, along with a linker, allowing equal expression of both chains. Using this retroviral vector, we successfully transduced this TCR into allogeneic peripheral blood mononuclear cells (PBMCs) and generated an antigen-specific TCR-T cell line (Figure 4). This TCR-T cell line recognized its cognate epitope and killed tumor cells expressing the COL6A3 antigen, but not negative control targets (Figures 5-6). Based on the current study, we plan to evaluate the safety and efficacy of this TCR-T therapy for the treatment of HLA-A*0201-positive patients with advanced or recurrent cancer in a non-randomized, dose-finding phase I / II trial. Based on previously obtained results, we will generate and expand TCR-T cells using the patient's own PBMCs. After functional detection (phenotype, killing ability, etc.), the TCR-T cells will be infused into the patient and safety will be evaluated using a dose-escalation strategy. If no severe dose-limiting toxicity (DTL) occurs, antitumor efficacy will be further assessed by staging evaluation and documented as complete response (CR), partial response (PR), or progressive disease (PD) (RECIST criteria). At this time, the duration of the transferred TCR-T will be determined. Progression-free survival, although not a primary endpoint of this phase I trial, will also be assessed.If no serious side effects are observed and a major clinical response is observed, we plan to apply a phase II trial to further evaluate the efficacy of this TCR-T cell in a larger cohort of HLA-A2 advanced cancer patients.

[0164] Table 1. Tumor targets used for functional validation of COL6A3-642-specific CTL cell lines TIFF0007827645000019.tif38160

[0165] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present invention. While the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein and in the steps or order of steps of the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

1. An engineered T cell receptor (TCR) that specifically binds to the human tumor antigen COL6A3, comprising a TCR-a polypeptide and a TCR-b polypeptide, the TCR-a polypeptide comprises CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 6-8, respectively; and the TCR-b polypeptide comprises CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 12-14, respectively; Engineered TCR.

2. The TCR of claim 1, wherein the TCR-a polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and the TCR-b polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

10.

3. The TCR of claim 1, wherein the TCR-a polypeptide comprises the amino acid sequence of SEQ ID NO:4 and the TCR-b polypeptide comprises the amino acid sequence of SEQ ID NO:

10.

4. (a) the TCR contains modifications or is chimeric; and / or (b) the TCR-a polypeptide and the TCR-b polypeptide are operably linked; A TCR according to any one of claims 1 to 3.

5. A TCR according to any one of claims 1 to 3 which is a single-chain TCR.

6. (a) the TCR-a polypeptide and the TCR-b polypeptide are on the same polypeptide, and the TCR-b is amino-proximal to the TCR-a; and / or (b) the TCR-a polypeptide and the TCR-b polypeptide are on the same polypeptide, and the TCR-a is amino-proximal to the TCR-b; and / or (c) the TCR comprises a linker between the TCR-a polypeptide and the TCR-b polypeptide; and / or (d) the linker comprises glycine and serine residues; A TCR according to any one of claims 1 to 3.

7. The TCR of any one of claims 1 to 3, conjugated to a detection agent or a therapeutic agent.

8. A fusion protein comprising the TCR of any one of claims 1 to 3 and a CD3 binding region, wherein the CD3 binding region comprises a CD3-specific fragment antigen-binding (Fab), a single-chain variable fragment (scFv), a single-domain antibody, or a single-chain antibody.

9. 10. The fusion protein of claim 8, conjugated to a detection agent or a therapeutic agent.

10. A nucleic acid encoding the TCR of any one of claims 1 to 3 or the fusion protein of claim 8 or 9.

11. 11. A nucleic acid vector comprising the nucleic acid of claim 10, wherein the nucleic acid vector comprises a promoter that directs expression of the nucleic acid.

12. A cell comprising a TCR according to any one of claims 1 to 3, a fusion protein according to claim 8 or 9, a nucleic acid according to claim 10, or a vector according to claim 11.

13. 13. The cell of claim 12, wherein the cell is a stem cell, a progenitor cell, an immune cell, a T cell, or a natural killer (NK) cell.

14. the cell is a hematopoietic stem or progenitor cell, a cell differentiated from a mesenchymal stem cell (MSC), an induced pluripotent stem cell (iPSC), a cell isolated from or derived from a peripheral blood mononuclear cell (PBMC), a cytotoxic T lymphocyte (CTL), a CD8+ T cell, a CD4+ T cell, an invariant NK T (iNKT) cell, a gamma-delta T cell, an NKT cell, or a regulatory T cell; and / or Cells are isolated from a cancer patient; The cell of claim 13.

15. 13. A composition for treating cancer in a subject, comprising the TCR of any one of claims 1 to 3, the fusion protein of claim 8 or 9, the nucleic acid of claim 10, or the vector of claim 11, or the cell of claim 12, wherein the composition has been determined to be one or more of serum-free, mycoplasma-free, endotoxin-free, and / or sterile.

16. 12. A method for producing an engineered cell or a population of engineered cells, comprising: transferring the nucleic acid of claim 10 or the vector of claim 11 into a cell to obtain the engineered cell; and further comprising one or more of the steps of culturing the engineered cell in a medium, screening the cell, and / or freezing the cell.

17. (a) the subject has previously undergone treatment for cancer; and / or (b) the method further comprises administering an additional therapeutic agent; and / or (c) the cancer comprises stage I, II, III, or IV cancer; and / or (d) the cancer includes metastatic and / or recurrent cancer; and / or (e) the cancer includes glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, glioblastoma, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, or melanoma cancer; and / or (f) the subject is HLA-A2 positive and / or has been tested as HLA-A2 positive and / or the subject is HLA-A0201 positive and / or has been tested as HLA-A0201 positive; 16. The composition of claim 15.

18. 18. The composition of claim 17, wherein the cancer is glioblastoma.

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