Peptides targeting the VCY antigen, engineered T cell receptors, and methods of use

Abstract

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

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 359,973, filed on Jul. 11, 2022, which is hereby incorporated by reference in its entirety.

[0002] This application includes a Sequence Listing in compliance with the ST.26 format, which is hereby incorporated by reference in its entirety. The Sequence Listing, created on Jul. 10, 2023, is named MDACP1336WO.xml and is 27,212 bytes in size.

[0003] I. Field of the Invention The present invention relates to the field of cancer therapy.

Background Art

[0004] II. Background Adoptive T cell therapy is one of the potentially powerful cancer treatments that genetically modifies natural T cells to be tumor-specific and enhances 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 are highly promising against tumors. The efficacy of TCRs depends on their interaction with peptide major histocompatibility complexes (pMHCs), which are complexes formed by peptides bound to MHCs. Intracellular antigens are cleaved into peptide chains and presented by MHC molecules to form pMHCs. Most cytoplasmic proteins expressed by class I MHC proteins are defective ribosomal translation products and are cleaved into peptide chains by proteolysis. These peptides then bind to class I MHC proteins 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 them to T cell receptors of class II MHC proteins derived from T cells. T cell receptors derived from T cells (which need to match the patient's human leukocyte antigen (HLA) alleles) recognize these pMHCs and cause the killing of cancer cells (human class I MHC proteins are expressed from three gene regions: HLA-A, HLA-B, and HLA-C, and human class II MHC proteins are also expressed from three gene regions: HLA-DR, HLA-DP, and HLA-DQ). Manipulation of TCRs directed against cancer-specific antigens and useful for cancer treatment is required. Summary of the Invention

[0005] The present disclosure provides peptides, engineered T cell receptors (TCRs), cells comprising peptides and TCRs, and methods of making and using peptides and TCRs that are useful for vaccination and other applications. The present disclosure relates to TCRs that specifically recognize VCY (variable charge Y-linked) CT antigens, such as peptides having the amino acid sequence of SSQPSPSGPK (SEQ ID NO:15).

[0006] Accordingly, the present disclosure describes polypeptides comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:8. Similarly provided are polypeptides comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:14.

[0007] The present disclosure similarly provides T cell receptors (TCRs) and engineered TCRs, such as a TCR comprising a TCR-α polypeptide and a TCR-β polypeptide, wherein the TCR-α polypeptide comprises a CDR3 comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:8, and the TCR-β polypeptide comprises a CDR3 comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:14. The TCR-α polypeptide may comprise a CDR3 comprising an amino acid sequence having 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) sequence identity with SEQ ID NO:8 or comprising an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO:8, and the TCR-β polypeptide may comprise a CDR3 comprising an amino acid sequence having 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) sequence identity with SEQ ID NO:14 or comprising an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity with SEQ ID NO:14.The TCR-α polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:8, and the TCR-β polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0008] Provided is a fusion protein comprising the TCR and CD3 binding region of the present disclosure. The CD3 binding region may comprise a CD3-specific fragment antigen binding (Fab), single-chain variable fragment (scFv), single domain antibody, or single-chain antibody. Exemplary CD3-specific fragment antigen binding (Fab) are known in the art. For example, US20180222981, which is incorporated herein by reference, discloses variable regions that specifically bind to CD3 that can be used in aspects of the present disclosure. Anti-CD3 antibodies and variable regions are disclosed in US20180117152, which is also incorporated by reference.

[0009] The present disclosure provides a peptide having at least 66% sequence identity with the peptide of SEQ ID NO:15. Also disclosed are polypeptides comprising the peptides of the present disclosure. The peptide or polypeptide may have 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) sequence identity with SEQ ID NO:15, or may have 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) sequence identity.

[0010] Also described are molecular complexes comprising a peptide or polypeptide of the disclosure and an MHC polypeptide. Methods include those for producing peptide-specific immune effector cells that include: (a) obtaining a starting population of immune effector cells; and (b) contacting the starting population of immune effector cells with a peptide of the disclosure, thereby generating peptide-specific immune effector cells. The disclosure also describes peptide-specific engineered T cells and TCRs produced by the methods of the disclosure. The disclosure also describes in vitro isolated dendritic cells comprising a peptide, polypeptide, nucleic acid, or expression vector of the disclosure.

[0011] Also provided are methods for prognosticating a patient or detecting a T cell response in a patient, the method including contacting a biological sample from the patient with a composition, peptide, or polypeptide of the disclosure. Included in this description are peptide-specific binding molecules that bind to a peptide of the disclosure or to a peptide-MHC complex. Exemplary binding molecules may or may not include antibodies, TCR mimicking antibodies, scFv, nanobodies, aptamers, and DARPINs. Related methods provide a method that includes contacting a composition comprising at least one MHC polypeptide and a peptide or polypeptide of the disclosure with a composition comprising T cells, and detecting T cells having a bound peptide and / or MHC polypeptide by detecting a detection tag.

[0012] Also provided is a kit comprising a peptide, polypeptide, nucleic acid, expression vector, or composition of the present disclosure. Methods include methods of cloning a T cell receptor (TCR), the method comprising: (a) obtaining a starting population of immune effector cells; (b) contacting the starting population of immune effector cells with a peptide of the present disclosure, thereby generating peptide-specific immune effector cells; (c) purifying the peptide-specific immune effector cells; and (d) isolating the TCR sequence from the purified immune effector cells. Also described is a method of generating a cell comprising introducing a nucleic acid or expression vector of the present disclosure into a cell. Methods include in vitro methods for generating a therapeutic T cell vaccine comprising co-culturing T cells with a peptide of the present disclosure.

[0013] The nucleic acids of the present disclosure include those encoding the CDR regions, variable regions, engineered TCRs, polypeptides, TCR-α polypeptides, TCR-β polypeptides, peptides, polypeptides, and fusion proteins described herein. The nucleic acid may be RNA. The nucleic acid may be DNA or cDNA encoding a peptide or polypeptide, or a complement of a peptide or polypeptide. The nucleic acid may include one of SEQ ID NO:1, 2, or a fragment thereof. The nucleic acid may have 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 with one of SEQ ID NO:1, 2, or a fragment thereof, or may include nucleotides 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. Also provided are compositions comprising a polypeptide, cell, nucleic acid, or engineered TCR of the present disclosure. Also described is a method of making an engineered cell comprising the step of introducing a nucleic acid or expression vector of the present disclosure into a cell. The methods include methods for treating cancer in a subject, comprising the step of administering a polypeptide, composition, cell, nucleic acid, or engineered TCR to a subject in need thereof. The methods include methods of reducing tumor burden; methods of lysing cancer cells; methods of killing tumor / cancer cells; methods of prolonging overall survival; methods of reducing the risk of cancer or tumor formation; methods of prolonging recurrence-free survival; methods of preventing cancer; and / or methods of reducing, eliminating, or decreasing the spread or metastasis of cancer, comprising the step of administering a polypeptide, composition, cell, nucleic acid, or engineered TCR to a subject in need thereof.

[0014] The polypeptide of the present disclosure or the TCR-α polypeptide may include a CDR3 having an amino acid sequence with 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 therefrom) 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 therefrom) sequence identity. The polypeptide may include a CDR3 that includes the amino acid sequence of SEQ ID NO:8. The polypeptide of the present disclosure or the TCR-β polypeptide may include a CDR3 having an amino acid sequence with 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 therefrom) sequence identity to SEQ ID NO:14, 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 therefrom) sequence identity. The polypeptide of the present disclosure or the TCR-β polypeptide may include a CDR3 that includes the amino acid sequence of SEQ ID NO:14. The engineered TCR may include a TCR-α polypeptide that includes a CDR3 having the amino acids of SEQ ID NO:8 and a TCR-β polypeptide that includes a CDR3 having the amino acid sequence of SEQ ID NO:14.

[0015] The polypeptide may comprise a variable region containing CDR1, CDR2, and CDR3 from a TCR-α polypeptide and / or a TCR-β polypeptide. The variable region may comprise a CDR1 having at least 80% sequence identity to SEQ ID NO:6. The variable region 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:6, or 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. The variable region may comprise a CDR2 having at least 80% sequence identity to SEQ ID NO:7. The variable region 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 may comprise 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. The variable region may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:6 and / or a CDR2 comprising 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.The variable region may include 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 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 variable region may include the amino acid sequence of SEQ ID NO:4. The polypeptide may include a T cell receptor alpha (TCR-a) variable region. The polypeptide may include a TCR-a variable region and a constant region. The polypeptide may further include a signal peptide. The signal peptide may include an amino acid sequence having at least 80% identity to SEQ ID NO:5. The signal peptide may include 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:5, 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. The signal peptide may include the amino acid sequence of SEQ ID NO:5. The variable region may include CDR1, CDR2, and / or CDR3.

[0016] The variable region may include a CDR1 having at least 80% sequence identity to SEQ ID NO:12. The variable region 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:12, or may include 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 variable region may include a CDR2 having at least 80% sequence identity to SEQ ID NO:13. The variable region 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 may include 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. The variable region may include 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. The variable region may include an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10.The variable region may include 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: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. The variable region may include the amino acid sequence of SEQ ID NO:10. The polypeptide may include a T cell receptor beta (TCR-b) variable region. The polypeptide may include a TCR-b variable region and a constant region. The polypeptide may or may further include a signal peptide. The signal peptide may include an amino acid sequence having at least 80% identity to SEQ ID NO:11. The signal peptide may include 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:11, 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. The signal peptide may include the amino acid sequence of SEQ ID NO:11. The variable region may include CDR1, CDR2, and / or CDR3.

[0017] The TCR can include a TCR-α polypeptide comprising a variable region containing CDR1, CDR2, and CDR3, and a TCR-β polypeptide comprising a variable region containing CDR1, CDR2, and CDR3. The TCR-α polypeptide can include a CDR1 having at least 80% sequence identity to SEQ ID NO:6, and / or the TCR-β polypeptide can include a CDR1 having at least 80% sequence identity to SEQ ID NO:12. The TCR-α polypeptide 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:6, or can include 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-β polypeptide 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:12, or can include 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-α polypeptide can include a CDR1 comprising the amino acid sequence of SEQ ID NO:6, and the TCR-β polypeptide can include a CDR1 comprising the amino acid sequence of SEQ ID NO:12. The TCR-α polypeptide can include a CDR2 having at least 80% sequence identity to SEQ ID NO:7, and the TCR-β polypeptide includes a CDR2 having at least 80% sequence identity to SEQ ID NO:13.The TCR-α polypeptide can have a sequence identity of 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) with respect to SEQ ID NO:7, or can contain a CDR2 having a sequence identity of 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) with respect to SEQ ID NO:7. The TCR-β polypeptide can have a sequence identity of 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) with respect to SEQ ID NO:13, or can contain a CDR2 having a sequence identity of 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) with respect to SEQ ID NO:13. The TCR-α polypeptide can contain a CDR2 containing the amino acid sequence of SEQ ID NO:7, and the TCR-β polypeptide can contain a CDR2 containing the amino acid sequence of SEQ ID NO:13. The CDR1, CDR2, and CDR3 of the TCR-α polypeptide can each contain the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and the CDR1, CDR3, and CDR3 of the TCR-β polypeptide can each contain the amino acid sequences of SEQ ID NOs:12, 13, and 14, respectively. The TCR-α variable region can contain an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO:4, and the TCR-β variable region can contain an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO:10.The TCR-α variable region can include 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, and the TCR-β variable region includes 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: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. The TCR-α polypeptide can include an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, and the TCR-β polypeptide includes an amino acid sequence having at least 70% sequence identity to SEQ ID NO:9.The TCR-a polypeptide can include 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, and the TCR-b polypeptide includes 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 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 TCR-a and / or TCR-b polypeptide can include a signal peptide. The signal peptide can include SEQ ID NO:5 or 11. The signal peptide can include 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:5 or 11, 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.

[0018] The TCR can be modified or chimeric. The variable region of the TCR can be fused to a TCR constant region that is different from the constant region of the cloned TCR that specifically binds to the peptides of the present disclosure.

[0019] The TCR-α polypeptide and the TCR-β polypeptide can be functionally linked. The term "functionally linked" can refer to a covalent bond such as a peptide bond (e.g., where the 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 having a certain degree of specific binding affinity for each other). The TCR-α polypeptide and the TCR-β polypeptide are functionally linked via a peptide bond. The TCR-α polypeptide and the TCR-β polypeptide are on the same polypeptide, and the TCR-β is amino-proximal to the TCR-α. The polypeptide can be further defined as a single-chain TCR. The TCR-α polypeptide and the TCR-β polypeptide may be on the same polypeptide, with the TCR-α being amino-proximal to the TCR-β. The TCR can include a linker between the TCR-α polypeptide and the TCR-β polypeptide. The linker can include glycine and serine residues. The linker can be composed of only glycine and serine residues (a glycine-serine linker). The linker can be a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n - SEQ ID NO:18, (G4S)n, and (GGGS)n - SEQ ID NO:19, where n is an integer of at least 1). n can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art can be used as the linker in the polypeptides of the present disclosure.Exemplary linkers can include, or consist of, GGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGSG (SEQ ID NO:22), GSGGG (SEQ ID NO:23), GGGSG (SEQ ID NO:24), GSSSG (SEQ ID NO:25), etc. Further linkers useful in the polypeptides and TCRs of the present disclosure are described herein. When the first region is attached to the carboxy terminus of the second region, the first region is carboxy-proximal to the second region. There may be additional amino acid residues intervening between the first and second regions. Thus, these regions need not be directly adjacent unless specifically specified as having no intervening amino acid residues. The term "amino-proximal" is similarly defined such that when the first region is attached to the amino terminus of the second region, the first region is amino-proximal to the second region. Similarly, there may be additional amino acid residues intervening between the first and second regions unless otherwise specified.

[0020] The CDRs can also include 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 one or both sides of a particular CDR sequence; thus, one or more additional amino acids can be present at the N-terminus or C-terminus of a particular CDR sequence, such as those shown in SEQ ID NOs: 6-8 and 12-14. Alternatively, or in combination, the CDRs can also be fragments of the CDRs described herein and can lack at least 1, 2, 3, 4, or 5 amino acids from the C-terminus or N-terminus of a particular CDR sequence.

[0021] 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. The TCR or fusion protein can be conjugated to the agents described herein.

[0022] The present disclosure also provides a nucleic acid encoding a TCR-α polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 and / or a TCR-β polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:14. The nucleic acid can have 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 can encode a TCR-α 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, and / or can have 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:14, or can encode a TCR-β 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 can encode a TCR-α polypeptide comprising CDR1, CDR2, and CDR3, and / or a TCR-β polypeptide comprising CDR1, CDR2, and CDR3. The nucleic acid can encode a TCR-α comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:6 and / or a TCR-β comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:12.The nucleic acid can encode a TCR-a having a CDR1 with 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 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 a TCR-b having a CDR1 with 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 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 nucleic acid can encode a TCR-a having a CDR2 with at least 80% sequence identity to SEQ ID NO:7 and / or a TCR-b having a CDR2 with at least 80% sequence identity to SEQ ID NO:13.The nucleic acid encodes a TCR-a having a sequence identity of 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) with respect to SEQ ID NO:7, or comprising a CDR2 having a sequence identity of 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) with respect to SEQ ID NO:7, and / or a TCR-b having a sequence identity of 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) with respect to SEQ ID NO:13, or comprising a CDR2 having a sequence identity of 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) with respect to SEQ ID NO:13. The nucleic acid can encode a TCR-a variable region comprising an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO:4 and / or a TCR-b variable region comprising an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO:10.The nucleic acid can encode 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 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, and / or 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 SEQ ID NO:10, 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. The nucleic acid can encode a TCR-a polypeptide chain comprising the amino acid sequence of SEQ ID NO:4 and / or a TCR-b chain comprising the amino acids of SEQ ID NO:10. The nucleic acid can comprise SEQ ID NO:1 and / or SEQ ID NO:2.The nucleic acid may include a nucleic acid sequence having a sequence identity of 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) with respect to SEQ ID NO:1, 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) of sequence identity, and / or having a sequence identity of 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) with respect to SEQ ID NO:2, 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) of sequence identity.

[0023] The nucleic acid may include TCR-a (TRA) and TCR-b (TRB) genes. The nucleic acid may be polycistronic. The nucleic acid may also include an internal ribosome entry site (IRES) or a P2A linker. The nucleic acid may include cDNA encoding the TCR-a and / or TCR-b gene. The nucleic acid may encode a polypeptide containing a CD3 binding region or may further encode it. The CD3 binding region may include a CD3-specific fragment antigen-binding (Fab), a single-chain variable fragment (scFv), a single-domain antibody, or a single-chain antibody.

[0024] The peptide may comprise at least 6 consecutive amino acids of the peptide of SEQ ID NO:15. The peptide may comprise 4, 5, 6, 7, 8, or 9 consecutive amino acids of the peptide of SEQ ID NO:15, may consist of 4, 5, 6, 7, 8, or 9 consecutive amino acids of the peptide of SEQ ID NO:15, or may comprise at least 4, 5, 6, 7, 8, or 9 consecutive amino acids of the peptide of SEQ ID NO:15. The peptide may comprise the amino acid sequence of SEQ ID NO:15 or may consist of the amino acid sequence of SEQ ID NO:15. The peptide may be 13 amino acids in length or shorter. The peptide may have at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any range derivable therein) amino acids, at most 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any range derivable therein) amino acids, exactly 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any range derivable therein) amino acids, or may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any range derivable therein) amino acids. The peptide may consist of 9 amino acids. The peptide may consist of 8 amino acids. The peptide may consist of 7 amino acids. The peptide may consist of 6 amino acids. The peptide may be immunogenic. The term immunogenic refers to the generation of an immune response such as a defensive immune response. The peptide may be modified. The modification may include conjugation to a molecule. The molecule may be an antibody, lipid, adjuvant, or detection moiety (tag). The peptide may comprise 100% sequence identity to the peptide of SEQ ID NO:15.The peptides of the present disclosure have 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to the peptide of SEQ ID NO:15, or those having at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity are also included. The peptide can have 77% sequence identity to the peptide of SEQ ID NO:15, or can have at least 77% sequence identity to the peptide of SEQ ID NO:15. The peptide can have 88% sequence identity to the peptide of SEQ ID NO:15, or can have at least 88% sequence identity to the peptide of SEQ ID NO:15. The peptides of the present disclosure can have 1, 2, or 3 substitutions relative to the peptide of SEQ ID NO:15. The peptide can have at least 1, 2, 3, 4 or 5 substitutions relative to the peptide of SEQ ID NO:15, or can have at most 1, 2, 3, 4 or 5 substitutions.

[0025] The composition can be formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. The peptide can be included in liposomes, lipid-containing nanoparticles, or lipid-based carriers. The composition of the present disclosure can be formulated as a vaccine. The composition can further contain an adjuvant.

[0026] Regarding the dendritic cells of the present disclosure, the dendritic cells can include mature dendritic cells. The cells can be cells having HLA-A type. HLA may include or exclude HLA-A, HLA-B, or HLA-C. The cells can be of HLA-A11 type. The cells can be of HLA-A*1101 type. The cells may include or exclude HLA-A01, HLA-A02, HLA-A11, HLA-A24, HLA-B07, HLA-B08, HLA-B15, or HLA-B40 plus cells.

[0027] The method can further include the step of isolating the expressed peptide or polypeptide. The T cells can include CD8+ T cells. The T cells may include or exclude CD4+ T cells, Th1, Th2, Th17, Th9, or Tfh T cells, cytotoxic T cells, memory T cells, central memory T cells, or effector memory T cells.

[0028] The vector can include both TCR-a and TCR-b genes. The vector can include a promoter that directs the expression of nucleic acids. The promoter can include the mouse stem cell virus (MSCV) promoter.

[0029] The cells may or may not include stem cells, progenitor cells, immune cells or natural killer (NK) cells. The cells may or may not include hematopoietic stem cells or hematopoietic progenitor cells, T cells, cells differentiated from mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs). The cells can be isolated or derived from peripheral blood mononuclear cells (PBMCs). The T cells may or may not 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. The cells can be isolated from cancer patients. The cells can be isolated from non-cancer patients. The cells can be isolated from healthy patients. The cells may or may not be frozen. The cells may be in cell culture. The cells may lack endogenous expression of the TCR gene. The cells may further include a chimeric antigen receptor (CAR). The cells may exclude the expression of the nucleic acid encoding the CAR or may exclude the nucleic acid encoding the CAR.

[0030] The composition may be determined to be serum-free, mycoplasma-free, endotoxin-free, and / or sterile. The method may, further may, or may not include the steps of culturing the cells in a medium, incubating the cells under conditions that allow cell division, screening the cells, and / or freezing the cells.

[0031] The subject may or may not include those diagnosed with cancer, such as the cancer described in this specification. The cancer may or may not include solid tumors. The subject may or may not include those who have been previously treated for cancer. The subject may be determined to be resistant to previous treatments. The method may or may not include administering further treatment. The cancer may be further defined as a solid tumor. The cancer may or may not include blood cancers, such as leukemia. The cancer may be the cancer described in this specification. The cancer may or may not include stage I, II, III, or IV cancer. The cancer may or may not include metastatic and / or recurrent cancer. The cancer may include VCY CT antigen+ cancer. The cancer may include cancer that expresses the peptide of SEQ ID NO:15. The subject or patient may or may not include those determined to have VCY CT antigen+ cancer cells or cancer cells positive for the epitope of SEQ ID NO:15. The cancer may be VCY CT antigen overexpressing cancer, such as overexpressing compared to non-cancerous subjects. The subject or patient may be determined to have VCY CT antigen overexpression in a biological sample. The biological sample may include cancer cells or a biopsy. The subject may be a mammal. The subject may include experimental animals, such as mice, rats, rabbits, dogs, cats, horses, or pigs. The subject may be a human. The subject may be determined to be positive for HLA-A11 and / or HLA-A*1101.

[0032] The compositions of the present disclosure may be formulated as vaccines. The compositions and methods of the present disclosure provide prophylactic therapy for preventing cancer. The compositions and methods of the present disclosure provide therapeutic therapy for treating existing cancer, such as for treating patients with cancer. The composition may or may further include an adjuvant. Adjuvants are known in the art and include, for example, TLR agonists and aluminum salts.

[0033] The method of the present disclosure may or may not include a step of screening cells for one or more cell characteristics, such as immunogenicity, including TCR expression, integration of nucleic acids encoding TCR genes, or binding of TCRs to cancer antigens such as VCY CT antigen or the peptide of SEQ ID NO:15.

[0034] The method may or may further include a step of administering a cell or a composition comprising the cell, wherein the cell includes autologous cells. The cell may include allogeneic cells. The cell may be allogeneic or xenogeneic.

[0035] The composition may contain the MHC polypeptide and peptide of the present disclosure, where the MHC polypeptide and / or peptide is conjugated to a detection tag. Thus, suitable detection tags include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins including enzymes. The tag may simply detect or may quantify. A response that is simply detected generally includes a response that simply confirms its presence, while a response that is quantified generally includes a response having a quantifiable (e.g., numerically reportable) value such as intensity, polarization, and / or other characteristics. In luminescence or fluorescence assay methods, the detectable response can occur directly using a luminophore or fluorophore associated with an assay component that actually participates in the binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent tags that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. Examples of suitable fluorescent tags include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methylcoumarin, pyrene, malachite 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 (6th). The detection tag also includes streptavidin or its binding partner biotin.

[0036] MHC polypeptides and peptides can be functionally linked. The term "functionally linked" refers to a situation where two components are combined or can be combined to form a complex. For example, the components may be covalently linked on the same polypeptide, such as in a fusion protein, and / or may be on the same polypeptide, or the components may have some degree of binding affinity for each other, such as the binding affinity resulting from van der Waals forces. MHC polypeptides and peptides can be functionally linked through a peptide bond. MHC polypeptides and peptides can be functionally linked through van der Waals forces. Peptide-MHC can be functionally linked to form a pMHC complex. At least two pMHC complexes can be functionally linked together. Similarly included are at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes that are functionally linked to each other, or at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes that are functionally linked to each other. At least two MHC polypeptides can be linked to one peptide. The average ratio of MHC polypeptide to peptide can be from 1:1 to 4:1. The ratio or average ratio can be at least 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any range derivable therein), at most 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any range derivable therein), or about 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any range derivable therein).

[0037] Peptides can be complexed with MHC. MHC can include HLA-A, HLA-B, or HLA-C types. Peptides can be loaded onto dendritic cells, lymphoblastoid cells, peripheral blood mononuclear cells (PBMCs), artificial antigen-presenting cells (aAPCs), or artificial antigen-presenting surfaces. The artificial antigen-presenting surface can include MHC polypeptides conjugated or linked to the surface. Exemplary surfaces include beads, microplates, slide glasses, or cell culture plates.

[0038] The method of the present disclosure may further include, or exclude, the step of counting the number of T cells bound to a peptide and / or MHC. The composition containing T cells can be isolated from a subject. The subject can be one defined herein, such as a human subject. The method may further include, or exclude, the step of sorting the number of T cells bound to a peptide and / or MHC. The method of the present disclosure may similarly include, or exclude, the step of sequencing one or more TCR genes from T cells bound to a peptide and / or MHC. The method may include, or exclude, the step of sequencing the TCRα gene and / or β gene from a TCR, such as a TCR that binds to a peptide of the present disclosure. The method may similarly include, or exclude, GLIPH (grouping of lymphocyte interactions by paratope hotspots) analysis. This is further described in Glanville et al., Nature. 2017 Jul 6; 547(7661): 94-98, which is incorporated herein by reference.

[0039] The composition of the present disclosure can be serum-free, mycoplasma-free, endotoxin-free, and / or sterile. The method may further include culturing the cells of the present disclosure in a medium, incubating the cells under conditions that allow cell division, screening the cells, and / or freezing the cells. The method may similarly further include isolating a peptide or polypeptide expressed from the cells of the present disclosure.

[0040] The method of the present disclosure may include, or further include, the step of screening dendritic cells for one or more cell characteristics. The method may further include contacting the cells with one or more cytokines or growth factors. The one or more cytokines or growth factors can include GM-CSF. The cell characteristics can include the cell surface expression of one or more of CD86, HLA, and CD14. The dendritic cells may be derived from CD34+ hematopoietic stem cells or progenitor cells.

[0041] Contacting in the method of the present disclosure can be further defined as co - culturing a starting population of antigen - presenting cells (APCs) that present peptides on their surface with immune effector cells. The APC can be a dendritic cell. The dendritic cell can be derived from peripheral blood mononuclear cells (PBMCs). The dendritic cell can be isolated from PBMCs. Cells from which the dendritic cell or DC is derived can be isolated by leukapheresis.

[0042] Peptide-MHC (pMHC) complexes can be prepared by contacting the peptides of the present disclosure with MHC complexes. The peptides can be expressed intracellularly and bind to endogenous MHC complexes to form pMHC. pMHC complexes can be prepared using peptide exchange. For example, cleavable peptides such as photocleavable peptides that bind to MHC and stabilize MHC may be designed. Cleavage of the peptide (e.g., by irradiation in the case of a photocleavable peptide) dissociates the peptide from the HLA complex, resulting in an empty HLA complex that rapidly decays unless UV irradiation is performed in the presence of a "rescue peptide". Thus, the peptides of the present disclosure can be used as "rescue peptides" in peptide exchange procedures. Similarly described are pMHC complexes comprising the peptides of the present disclosure. The pMHC complexes may be functionally linked to a solid support or attached to a detectable moiety such as a fluorescent molecule, a radioisotope, or an antibody. Similarly described are peptide-MHC multimer complexes comprising at least 1, 2, 3, 4, 5, or 6 peptide-MHC molecules, comprising 1, 2, 3, 4, 5, or 6 peptide-MHC molecules that are all functionally linked together, or comprising at most 1, 2, 3, 4, 5, or 6 peptide-MHC molecules. The linkage may be a covalent bond such as through a peptide bond or a non-covalent bond. The pMHC molecules may be bound to biotin molecules. Such pMHC molecules can be multimerized by binding to streptavidin molecules. The pMHC multimers can be used to detect antigen-specific T cells or TCR molecules present in a composition or tissue. The multimers can be used to detect peptide- or peptide-specific T cells in situ or in a biopsy sample. The multimers may be bound to a solid support or deposited on a solid support such as an array or a slide. Cells are then added to the slide, and binding between the pMHC multimers and the cells can be detected. Thus, the pMHC molecules and multimers of the present disclosure can be used to detect and diagnose cancer in a subject or to determine the immune response in an individual having cancer.

[0043] As defined in the methods described herein, obtaining may include, or exclude, isolating a starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs). The starting population of immune effector cells can be obtained from a subject. The methods of the present disclosure may include, or exclude, the step of introducing a peptide or a nucleic acid encoding a peptide into dendritic cells prior to co-culture. Introduction of the peptide can be effected by transfecting or infecting dendritic cells with a nucleic acid encoding the peptide, or by incubating the peptide with dendritic cells. The peptide or nucleic acid encoding a peptide can be introduced by electroporation. Other methods of nucleic acid transfer, such as lipofection, calcium phosphate transfection, transfection with DEAE-dextran, microinjection, and virus-mediated transduction, are known in the art and useful in the methods of the present disclosure for transferring the nucleic acids of the present disclosure into cells, or may be excluded in the methods described herein. The peptide or nucleic acid encoding a peptide can be introduced by adding the peptide or nucleic acid encoding a peptide to the dendritic cell culture medium. The immune effector cells can be co-cultured with a second population of dendritic cells into which the peptide or nucleic acid encoding a peptide has been introduced. After co-culture, a population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells can be purified from the immune effector cells. The population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells can be purified by fluorescence-activated cell sorting (FACS). A clonal population of peptide-specific immune effector cells can be generated by limiting dilution or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

[0044] Purification may further include generating a clonal population of peptide-specific immune effector cells by limiting dilution or serial dilution of the selected cells followed by expansion of individual clones by a rapid expansion protocol. The methods of the present disclosure may include, or further include, cloning of the T cell receptor (TCR) from a clonal population of peptide-specific immune effector cells. The term isolation in the methods of the present disclosure may be defined as, or further defined as, cloning of the T cell receptor (TCR) from a clonal population of peptide-specific immune effector cells. Cloning of the TCR may include cloning of the TCRα and β chains. The TCR may be cloned using the 5'-cDNA end rapid amplification (RACE) method. The TCRα and β chains may be cloned using the 5'-cDNA end rapid amplification (RACE) method. The cloned TCR may be subcloned into an expression vector. The expression vector may include a linker domain between the TCRα and TCRβ sequences. The expression vector may be a retroviral vector or a lentiviral vector. The vector may be an expression vector described herein. The linker domain may include a sequence encoding one or more peptide cleavage sites. The one or more cleavage sites may be a furin cleavage site and / or a P2A cleavage site. The TCRα and TCRβ sequences may be linked by an IRES sequence.

[0045] To generate engineered cells that express the TCRα and / or β chains, the host cells of the present disclosure may be transduced with an expression vector. The host cells may be immune cells. The immune cells may be T cells, and the engineered cells may be referred to as engineered T cells. The T cells may be CD8 +It may be a type of T cell as described herein, such as a T cell, a CD4+ T cell, or a γδ T cell. The starting population of immune effector cells may be obtained from a subject having cancer, and the host cells may be allogeneic or autologous to the subject. The peptide-specific T cells may be autologous or allogeneic. A population of engineered T cells that are CD4-positive or CD8-positive and peptide MHC tetramer-positive can be purified from the transduced host cells. A clonal population of peptide-specific engineered T cells can be generated by limiting dilution or serial dilution, followed by expansion of individual clones by a rapid expansion protocol. In the methods of the present disclosure, purification can be defined as purifying a population of CD4-positive or CD8-positive and peptide MHC tetramer-positive T cells from immune effector cells after co-culture.

[0046] The peptide can be linked to a solid support. The peptide can be conjugated to a solid support or bound to an antibody conjugated to a solid support. The solid support can include a microplate, beads, a glass surface, a slide, or a cell culture dish. The solid support can include a nanofluidic chip. Detecting a T cell response can include or further include detecting the binding of the peptide to the T cell or TCR. Detecting a T cell response can include an ELISA, an ELISPOT, or a tetramer assay.

[0047] The methods of the present disclosure can also be used to determine the effectiveness of a vaccine, such as a cancer vaccine.

[0048] "Treatment" or "treating" refers to any treatment of a disease in a mammal, including (i) preventing the disease, i.e., not causing the clinical symptoms of the disease by administering a protective composition before the induction of the disease; (ii) suppressing the disease, i.e., not causing the clinical symptoms of the disease by administering a protective composition after an inducing event but before the clinical appearance or reappearance of the disease; (iii) arresting the disease, i.e., preventing the onset of clinical symptoms by administering a protective composition after its first appearance; and / or (iv) alleviating the disease, i.e., causing regression of the clinical symptoms by administering a protective composition after its first appearance. Treatment may exclude prevention of the disease.

[0049] Throughout this application, the term "about" is used in its plain and ordinary sense in the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the apparatus or method used to determine that value.

[0050] 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 two or more".

[0051] As used herein, the terms "or" and "and / or" are used to combine or describe mutually exclusive of a plurality of components. For example, "x, y, and / or z" can mean "x only", "y only", "z only", "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.

[0052] The terms "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 non - limiting and do not exclude additional, unrecited elements or method steps.

[0053] Compositions and methods for its use can "comprise", "consist essentially of" or "consist of" any of the components or steps disclosed throughout this application. The phrase "consist of" excludes any element, step, or component not specified. The phrase "consist essentially of" limits the scope of the described subject matter to those that do not materially affect the basic and novel characteristics of the particular materials or steps. Aspects and embodiments described in the context of the term "comprise" are also contemplated to be practicable in the context of the terms "consist of" or "consist essentially of".

[0054] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect can also be described in the language of a "use" claim, such as "the use of" any compound, composition, or agent discussed herein, to achieve or effect the described therapeutic, diagnostic, or physiological purpose or effect.

[0055] The use of one or more arrays or compositions can be utilized based on any of the methods described herein. Other aspects are discussed throughout this application. Any aspect or feature discussed with respect to one aspect of the present disclosure is equally applicable to other aspects of the present disclosure, and vice versa.

[0056] It is particularly contemplated that any limitation discussed with respect to one aspect or feature of the present invention may be applicable to any other aspect or feature of the present invention. Further, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention. The aspects and features described in the examples may also be implemented in the context of aspects discussed elsewhere in different examples or elsewhere in this application, such as in the summary of the invention, the detailed description of the aspects, the claims, and the description of the legends of the figures.

[0057] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples indicate certain aspects and features of the present invention, it is to be understood that the detailed description is by way of illustration only and that various modifications and changes within the spirit and scope of the present invention will be apparent to those skilled in the art.

Brief Description of the Drawings

[0058] The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to these drawings in conjunction with the detailed description of the specific aspects presented herein.

[0059] (Figure 1) MS identification of VCY HLA-A1101-restricted peptide. MHC / peptide was isolated from the lysate of K562-A11-VCY cell line using immunoprecipitation (IP). The peptide was eluted using acetic acid. The eluted peptide was separated by HPLC and identified by mass spectrometry (MS). One HLA-A1101-restricted peptide VCY-23 (SSQPSPSGPK - SEQ ID NO:15) was found to have a high ion score (ion score = 34). (Figure 2-1) VCY-23 peptide SSQPSPSGPK (SEQ ID NO:15) MS-Western blot assay. Isotope-labeled VCY-23 peptide (heavy peptide) was spiked into the MHC / peptide eluate from K562-A11-VCY cell line. Then, target-MS specific for VCY-23 peptide was performed. Both native VCY-23 peptide (Figure 1) and heavy peptide (Figure 2) were found and identified. These results indicated that VCY-23 peptide was naturally processed and presented in K562-A11-VCY cell line. (Figure 2-2) Refer to the description of Figure 2-1. (Figure 3) Generation of VCY-23 HLA-A1101-restricted peptide (SSQPSPSGPK - SEQ ID NO:15) CTL. VCY-23 (SSQPSPSGPK - SEQ ID NO:15) peptide was pulsed into mature dendritic cells and then co-cultured with autologous PBMC of HLA-A1101+ healthy donors. After two rounds of stimulation, a part of T cells was collected from each well for dextramer detection. Then, the tetramer+ / CD8+ population was sorted and expanded by rapid expansion protocol (REP). After 2 weeks of REP, high-purity CTL (more than 90% tetramer+ population) was observed. (Fig. 4-1) Figs. 4A - F: Functional verification of VCY-23 peptide-specific CTL cell lines. (A) K562-A11 cells pulsed with various concentrations of VCY-23 peptide were used as targets. The lytic ability of the VCY-23 CTL cell line was detected by the Cr51 release assay (CRA). The effector-to-target (E:T) ratio was 20:1. (B) K562-A11 (A1101+, VCY-), K562-A11-VCY (A1101+, VCY+), and K562-A2-VCY (A1101-, VCY+) were used as targets to test the killing ability of VCY-23 CTL using CRA. (C - F) Tumor cell lines H647, M14, H1299-A11, and H2023-A11 (left), as well as VCY-overexpressing cell lines (right) (all A1101+) were used as targets to test the recognition of the VCY-23 CTL cell line using CRA. (Fig. 4-2) See the description of Fig. 4-1. (Fig. 4-3) See the description of Fig. 4-1. (Fig. 5) Non-radioactive target inhibition assay to verify the recognition specificity of VCY-23-specific CTL. VCY-overexpressing tumor cell lines labeled with 51Cr were used as radioactive targets. K562-A11 cells pulsed with VCY-23 peptide without 51Cr labeling were used as non-radioactive targets. K562-A11 cells pulsed with irrelevant peptides were used as control non-radioactive targets. E:T was 20:1. Non-radioactive target:Radioactive target was 10:1 or 20:1. Inhibition of killing of non-radioactive targets against VCY-23 CTL was detected by CRA. (Fig. 6) Cross-reactivity assay of VCY-23 peptide-specific CTL cell lines. VCX-overexpressing K562-A11 cells, and various concentrations of VCX-23 peptide K562 - A11 cells pulsed with TIFF2025523003000001.tif4128 were used as targets. The cytotoxicity of the VCY - 23 CTL cell line was detected by a Cr51 release assay (CRA). As a result, since VCY - 23 CTL did not kill the VCX - overexpressing K562 - A11 cell line and the K562 - A11 cell line pulsed with the VCX - 23 peptide, it was shown that VCY - 23 CTL does not cross - recognize the similar peptide VCX - 23 which differs from the VCY - 23 peptide by only one amino acid. (Figure 7) Generation of VCY - 23 - specific TCR - T. The full - length TCR was cloned into the retroviral vector pMSGV1 to generate a recombinant retrovirus. PBMCs from HLA - A1101 healthy donors were infected with the recombinant retrovirus. After infection, the tetramer + / CD8+ or tetramer + / CD4+ populations were separately sorted and expanded. After expansion, high - purity VCY - 23 - specific TCR - T was obtained. (Figure 8 - 1) Figures 8A - F: Functional verification of VCY - 23 - specific TCR - T by a killing assay. (A) K562 - A11 cells pulsed with various concentrations of the VCY - 23 peptide were used as targets. The cytotoxicity of CD8+ (left) or CD4+ (right) VCY - 23 TCR - T was detected by a Cr51 release assay (CRA). The effector - to - target (E:T) ratio used was 20:1. (B - F) The VCX - overexpressing cell line and the parental cell line were used as targets to test the recognition by CD8+ (left) or CD4+ (right) VCY - 23 TCR - T by CRA. The effector - to - target (E:T) ratios used were from 40:1 to 1.25:1. (Figure 8 - 2) Refer to the description of Figure 8 - 1. (Figure 8 - 3) Refer to the description of Figure 8 - 1. (Figure 9 - 1) Functional detection of VCY - 23 TCR - T by an intracellular cytokine staining (ICS) assay. CD8+ or CD4+ VCY - 23 TCR - T cell lines were co - cultured with the VCX - overexpressing cell line and the parental cell line at an E:T = 10:1 ratio. After overnight co - culture, the TCR - pathway downstream activation markers CD137, CD69, IFN - γ, and TNF - α were detected by an ICS assay. (FIG. 9-2) Refer to the description of FIG. 9-1. (FIG. 9-3) Refer to the description of FIG. 9-1. (FIG. 9-4) Refer to the description of FIG. 9-1.

DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed Description of the Invention The present disclosure provides a T cell receptor (TCR) that recognizes an HLA-A11-restricted epitope derived from the cancer testis (CT) antigen VCY having the amino acid sequence SSQPSPSGPK (SEQ ID NO: 15). The present invention similarly provides a nucleotide sequence encoding this TCR and an expression vector containing this nucleotide sequence that can be used to modify PBMCs to generate VCY-specific T cells. The present invention similarly provides the use of VCY-specific T cells for adoptive immunotherapy for HLA-A11 plus cancer patients in which malignant cells express the VCY antigen.

[0061] I. Engineered T Cell Receptor T cell receptors consist of two different polypeptide chains, designated the T cell receptor α (TCRα) chain and β (TCRβ) chain, which are linked by disulfide bonds. These α:β heterodimers are very similar in structure to the Fab fragment of an immunoglobulin molecule and are responsible for antigen recognition by most T cells. A minority of T cells have another, but structurally similar, receptor composed of pairs of different polypeptide chains called γ and δ. Both types of T cell receptors are different from the membrane-bound immunoglobulin that functions as a B cell receptor: T cell receptors have only one antigen-binding site, while B cell receptors have two, and T cell receptors are never secreted, while immunoglobulins can be secreted as antibodies.

[0062] Both chains of the T cell receptor have an amino-terminal variable (V) region that is homologous to the immunoglobulin V domain, a constant (C) region that is homologous to the immunoglobulin C domain, 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 ends in a short cytoplasmic tail.

[0063] The three-dimensional structure of the T cell receptor has been determined. This structure is indeed similar to that of the antibody Fab fragment, as predicted from previous studies on the genes encoding the T cell receptor. The chains of the T cell receptor are folded in much the same way as those of the Fab fragment, but the final structure appears to be somewhat 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, whose fold is different from that of any other immunoglobulin-like domain. Half of the domain juxtaposed to the Cβ domain forms a β-sheet similar to that found in other immunoglobulin-like domains, but the other half of the domain is formed by loosely packed strands and short segments of α-helix. Intramolecular disulfide bonds usually link two β-strands in immunoglobulin-like domains, but in the Cα domain, they link a β-strand to this segment of α-helix.

[0064] There are also differences in how the domains interact. The interface between the V and C domains of both T cell receptor chains extends over a wider area than in antibodies, which can reduce the mobility of the hinge bond between the domains. And the interaction between the Cα and Cβ domains is characterized in that the carbohydrate supports it by the sugar groups from the Cα domain making several hydrogen bonds to the Cβ domain. Finally, from a comparison of the variable binding sites, it is shown that the complementarity-determining region (CDR) loops are fairly closely aligned with the loops of the antibody molecule, but there are some substitutions compared to the loops of the antibody molecule. This substitution is particularly prominent in the Vα CDR2 loop and is oriented almost at right angles to the equivalent loop of the antibody V domain as a result of a shift in the β-strand that anchors one end of the loop from one side of the domain to the other. The chain substitution also causes a change in the orientation of the Vβ CDR2 loops in two of the seven Vβ domains whose structures are known. So far, the crystal structures of seven T cell receptors have been elucidated at this level of resolution.

[0065] Described herein are engineered T cell receptors. The term "engineered" refers to a T cell receptor having a TCR variable region grafted onto a TCR constant region to create a chimeric polypeptide that binds to the peptides and antigens of the present disclosure. The TCR is used for cloning, enhancing expression, detection, or for the therapeutic control of constructs, but contains intervening sequences not present in the endogenous TCR, such as multicloning sites, linkers, hinge sequences, modified hinge sequences, modified transmembrane sequences, detection polypeptides or molecules, or therapeutic controls that can enable the selection or screening of cells containing the TCR.

[0066] The TCR can include non-TCR sequences. Thus, the present disclosure describes TCRs having sequences not derived from TCR genes. The TCR can be chimeric in that it includes sequences from at least two TCR genes that are normally found in TCR genes but are not necessarily found together in nature.

[0067] The engineered TCRs of the present disclosure can include variable elements as shown below: TIFF2025523003000002.tif216159TIFF2025523003000003.tif239159

[0068] The following table relates to the characteristics of TCR-a: TIFF2025523003000004.tif57160

[0069] The following table relates to the characteristics of TCR-b: TIFF2025523003000005.tif74160

[0070] II. Proteinaceous Compositions As used herein, "protein", "peptide" or "polypeptide" refers to a molecule containing at least 5 amino acid residues. As used herein, the term "wild-type" refers to the endogenous version of a molecule that occurs naturally in an organism. A wild-type version of a protein or polypeptide may be utilized, however, a protein or polypeptide modified to elicit an immune response may also be utilized. 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 modified relative to the wild-type protein or polypeptide. A modified / variant protein or polypeptide may have at least one modified activity or function (recognizing that a protein or polypeptide can have multiple activities or functions). A modified / variant protein or polypeptide may be modified with respect to one activity or function but retain wild-type activity or function in other respects such as immunogenicity.

[0071] When a protein is specifically referred to in this specification, it generally refers to a native (wild-type) or recombinant (modified) protein or, optionally, a protein with any signal sequence removed. The protein can be isolated directly from the organism from which it is native, produced by recombinant DNA / heterologous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. Isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof) are included in the present disclosure. The term "recombinant" can be used in combination with the name of a polypeptide or a particular polypeptide, which generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a replication product of such a molecule.

[0072] The size of the protein or polypeptide (wild-type or modified) can be 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 residues or nucleic acid residues or more, and any range derivable therefrom, or derivatives of the corresponding amino sequences described or referenced herein, but is not limited thereto. The polypeptide may be mutated by truncation and be shorter than its corresponding wild-type form, and it is contemplated that it can be modified by fusing or conjugating a heterologous protein or polypeptide sequence to a particular function (e.g., for target targeting or localization, for enhancing immunogenicity, for purification purposes, etc.).

[0073] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the present disclosure may include, or exclude, 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, and / or of SEQ ID NO: 1-27, 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 therefrom, or At most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids, or any range derivable therefrom, and 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) may be similar, identical, or homologous. The peptide or polypeptide may be a human sequence or may be based on a human sequence. The peptide or polypeptide may not be naturally occurring and / or may be in a combination of peptides or polypeptides.,

[0074] The substitution or mutation is at any of amino acid positions 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, 268269、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、Substitutions or mutations at 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 or 650 (or any range derivable therefrom) may be included or excluded, may be substitutions with any amino acid, or may exclude substitutions with any amino acid, or, Substitutions with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine may be included or excluded.

[0075] The protein, polypeptide or nucleic acid is from amino acid or nucleotide 1 to 256 of SEQ ID NOs: 1 to 27, including 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, 256It may include or exclude 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 therefrom), or may exclude them.

[0076] The protein, polypeptide or nucleic acid is from amino acid or nucleotide 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264 of SEQ ID NO:1 - 27It may include or exclude 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), and has sequence identity of 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) with respect to one of SEQ ID NOs: 1 to 27, or has at least sequence identity of 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).

[0077] The protein, polypeptide or nucleic acid is of SEQ ID NOs: 1 to 27 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, and may contain consecutive amino acids or nucleic acids in (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, 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, 276277, 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, and may contain consecutive amino acids or nucleic acids in (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, 276277, 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, optionally contain consecutive amino acids or nucleic acids (or any range derivable therein), or 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) of consecutive amino acids or nucleic acids may be excluded.

[0078] The polypeptide, protein or nucleic acid is 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), 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 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 SEQ ID NOs: 1 - 27 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, 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, 276277, 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 therefrom), 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, 276277, 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 therefrom), or, Exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 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, 276It may contain 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 consecutive amino acids (or any range derivable therefrom).

[0079] The nucleic acid molecule or polypeptide is any of SEQ ID NOs: 1 to 27, 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、starting at position 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, and any of SEQ ID NO:1 to 27, 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, 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, 276277、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 therefrom), 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, 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, 276277、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 therefrom), 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, 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, 276277、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), and may contain a nucleic acid molecule or polypeptide comprising consecutive amino acids or nucleotides.

[0080] The nucleotide as well as 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 Genbank and GenPept databases of the National Center for Biotechnology Information (world wide web at ncbi.nlm.nih.gov / ) and the Universal Protein Resource (UniProt; world wide web at uniprot.org). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art.

[0081] In the compositions of the present disclosure, it is contemplated that there are 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 can 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).

[0082] The following is a consideration regarding changing the amino acid subunits of a protein to produce equivalent or in some cases improved, second-generation variant polypeptides or peptides. For example, a particular amino acid can be used in place of other amino acids in a protein or polypeptide sequence such that it is or is not accompanied by a substantial loss of the ability to interact and bind to a structure such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. Since the functional activity of a protein is defined by its ability to interact and its properties, certain amino acid substitutions can be made in the protein sequence and in its corresponding DNA coding sequence such that a protein with similar or desirable properties can still be produced. Thus, the inventors contemplate that various changes can be made to the DNA sequence of a gene encoding a protein without significantly losing its biological utility or activity.

[0083] The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations" that refer to changes in codons that encode biologically equivalent amino acids.

[0084] The amino acid sequence variants of the present disclosure can be substitution variants, insertion variants, or deletion variants. Mutations in the polypeptides of the present 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 therefrom) non - contiguous or contiguous amino acids of the protein or polypeptide as 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 referred to 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.

[0085] Amino acid and nucleic acid sequences can each contain additional N - terminal or C - terminal amino acids, or additional residues such as 5' or 3' sequences, so long as they meet the above - mentioned criteria including the maintenance of biological protein activity in which protein expression is involved, and it is also understood and considered that they can still be essentially identical as described for one of the sequences disclosed herein. The addition of terminal sequences applies particularly to nucleic acid sequences and can include various non - coding sequences adjacent to either the 5' or 3' portion of the coding region, for example.

[0086] Deletion variants typically lack one or more residues of the native or wild - type protein. Individual residues can be deleted, or several adjacent amino acids can be deleted. A stop codon can be introduced (by substitution or insertion) into the coding nucleic acid sequence to produce a truncated protein.

[0087] Insertion variants typically involve the addition of amino acid residues at non-terminal points in a polypeptide. This can include the insertion of one or more amino acid residues. It is also possible to create terminal adducts, which can include fusion proteins that are multimers or aggregates of one or more peptides or polypeptides described or referenced herein.

[0088] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and are designed to modify one or more properties of the polypeptide without losing or with only losing other functions or properties. The substitution may be conservative, i.e., one amino acid may be substituted for an amino acid with similar chemical properties. "Conservative amino acid substitutions" can 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, the change of 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 reverse or inverted amino acid moieties.

[0089] Alternatively, the substitution may be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting one amino acid residue with a chemically different residue, such as using a polar or charged amino acid in place of a non-polar or uncharged amino acid and vice versa. Non-conservative substitutions can involve the exchange of a member of one amino acid class with a member from another class.

[0090] One of ordinary skill in the art can determine suitable variants of the polypeptides as described herein using well-known techniques. One of ordinary skill in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions that are not thought to be important for activity. One of ordinary skill in the art will also be able to identify amino acid residues and portions of the molecule that are conserved among similar proteins or polypeptides. Regions that may be important for biological activity or structure can tolerate conservative amino acid substitutions without significantly changing biological activity or adversely affecting protein or polypeptide structure.

[0091] When making such changes, the hydropathy index of the amino acid can be considered. The hydropathy profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then repeatedly averaging these values along the peptide chain. Each amino acid has a value assigned based on its hydrophobic 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 hydropathy amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). The relative hydropathy characteristics of amino acids contribute to the secondary structure of the resulting protein or polypeptide, and as a result, it is accepted that this defines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that a particular amino acid can be substituted with other amino acids having similar hydropathy indices or scores and still retain similar biological activity. When making changes based on the hydropathy index, substitutions of amino acids with hydropathy indices within ±2 are included. In some aspects of the present invention, those within ±1 are included, and in other aspects of the present invention, those within ±0.5 are included.

[0092] It is also understood in the art that amino acids can be effectively substituted based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, is correlated with the biological properties of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, is correlated with its immunogenicity and antigen binding, i.e., with the biological properties of the protein. The following hydrophilicity values are assigned to these amino acid residues: 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). When making changes based on similar hydrophilicity values, in certain aspects, substitutions of amino acids with hydrophilicity values within ±2 are included, in other aspects, those within ±1 are included, and in still other aspects, those within ±0.5 are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions." Amino acids are understood to be able to be substituted with another amino acid having a similar hydrophilicity value and still result in a biologically equivalent and immunologically equivalent protein.

[0093] Furthermore, one of ordinary skill in the art can reevaluate structure - function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. From the perspective of such comparisons, the importance of amino acid residues in a protein corresponding to amino acid residues important for the activity or structure of a similar protein can be predicted. One of ordinary skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0094] One of ordinary skill in the art can also analyze the three - dimensional structure and amino acid sequence in relation to its structure in similar proteins or polypeptides. Taking such information into account, one of ordinary skill in the art can predict the alignment of the amino acid residues of the antibody with respect to its three - dimensional structure. For amino acid residues predicted to be on the surface of the protein, one of ordinary skill in the art may choose not to make changes because such residues may be involved in important interactions with other molecules. Furthermore, one of ordinary skill in the art can create test variants that contain a single amino acid substitution at each desired amino acid residue position. These variants can then be screened using standard assay methods for binding and / or activity, and thus information collected from such routine experiments can be obtained, whereby one of ordinary skill in the art can determine the amino acid positions at which further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found at expasy.org / proteomics / protein_structure on the World Wide Web.

[0095] (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) modifying the binding affinity for forming protein complexes, (4) modifying ligand or antigen binding affinity, and / or (5) imparting or modifying 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 a naturally occurring sequence. Substitutions can be made in that portion of the antibody outside the domain that forms intermolecular contacts. Conservative amino acid substitutions that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more substituted amino acids that do not disrupt the secondary structure characterizing a natural antibody) can be used.

[0096] III. Nucleic Acids Nucleic acid sequences can exist in a variety of examples such as: single-stranded or double-stranded of an antibody, or a fragment thereof, encoding a derivative, mutein or variant, an isolated segment of an incorporated sequence or recombinant polynucleotide and recombinant vector, a polynucleotide sufficient to be used as a hybridization probe, PCR primer or sequencing primer to identify, analyze, mutate or amplify a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting the expression of a polynucleotide, and the aforementioned complementary sequences described herein. Nucleic acids encoding epitopes for certain antibodies provided herein are also provided. Nucleic acids encoding fusion proteins containing these peptides are also provided. The nucleic acid can be single-stranded or double-stranded and can contain RNA and / or DNA nucleotides as well as their artificial variants (e.g., peptide nucleic acids).

[0097] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from whole genomic nucleic acids. Included within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids having a length of 100 residues or less), such as recombinant vectors including plasmids, cosmids, phages, viruses, and the like. A polynucleotide can include regulatory sequences that are substantially isolated from natural genes or protein coding sequences. A polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present within the polynucleotide.

[0098] In this regard, the terms "gene", "polynucleotide", or "nucleic acid" are used to refer to a nucleic acid that encodes a protein, polypeptide or peptide (including any sequences required for proper transcription, post-translational modification or localization). As will be understood by those skilled in the art, the term encompasses genomic sequences, expression cassettes, cDNA sequences, as well as genetically engineered smaller nucleic acid segments capable of expressing or being adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. Nucleic acids encoding all or part of a polypeptide can include contiguous nucleic acid sequences encoding all or part of such polypeptide. It is also contemplated that a particular polypeptide can be encoded by nucleic acids including variants that have slightly different nucleic acid sequences but still encode the same or substantially similar proteins.

[0099] Also included herein are polynucleotide variants having substantial identity with the sequences disclosed herein; said variants having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values and ranges therebetween, compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis with standard parameters). An isolated polynucleotide is a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95%, and more identity to the amino acid sequences described herein over the full length of the sequence; or may include a nucleotide sequence complementary to said isolated polynucleotide.

[0100] The nucleic acid segment, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and thus its full 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, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. Therefore, nucleic acid fragments of almost all lengths can be used, and it is preferably considered that its full length is restricted by the ease of purification and the use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence having additional heterologous coding sequences, for example, to enable purification, transport, secretion, post-translational modification of the polypeptide, or to enable therapeutic utility such as target targeting or efficacy. As discussed previously, a tag or other heterologous polypeptide can be added to the sequence encoding the modified polypeptide, and "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0101] A. Hybridization A nucleic acid that hybridizes to other nucleic acids under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, under moderately stringent hybridization conditions, a pre-washing solution containing 5× sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of approximately 50% formamide, 6× SSC, and a hybridization temperature of 55° C. (or other similar hybridization solutions such as those using a hybridization temperature of 42° C. and containing approximately 50% formamide), and washing conditions of 60° C. in 0.5× SSC, 0.1% SDS are used. Under stringent hybridization conditions, hybridization is carried out in 6× SSC at 45° C., followed by one or more washes in 0.1× SSC, 0.2% SDS at 68° C. Furthermore, one of ordinary skill in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids containing nucleotide sequences that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other generally remain hybridized to each other.

[0102] Parameters that affect the choice of hybridization conditions and guidance for devising appropriate conditions are described, for example, in Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated herein by reference in their entirety for all purposes) and can be readily determined by one of ordinary skill in the art, for example, based on the length and / or base composition of the DNA.

[0103] B. Mutations Mutations can be introduced into the nucleic acid, thereby resulting in changes in the amino acid sequence of the polypeptide encoded by the nucleic acid (e.g., an antibody or antibody derivative). Mutations can be introduced using any technique known in the art. One or more specific amino acid residues can be changed, for example, using site-directed mutagenesis protocols. One or more randomly selected residues can be changed, for example, using random mutagenesis protocols. However made, the mutant polypeptide can be expressed and screened for the desired properties.

[0104] The mutation can be introduced into the nucleic acid without significantly changing the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions can be made that result in amino acid substitutions at positions of non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of the encoded polypeptide. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutation can change the biological activity quantitatively or qualitatively. Examples of quantitative changes include an increase, decrease or elimination of activity. Examples of qualitative changes include a modification of the antigen specificity of an antibody.

[0105] 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 only a portion of the nucleic acid sequence encoding the full-length polypeptide, for example, a fragment that can be used as a probe or primer, or a fragment encoding only the active portion of a given polypeptide.

[0106] In another aspect, the nucleic acid molecule can be used as a probe or PCR primer for a specific antibody sequence. For example, the nucleic acid molecule probe can be used in a diagnostic method or, in particular, the nucleic acid molecule PCR primer can be used to amplify a region of DNA that can be used to isolate the nucleic acid sequence for use in the production of the variable domain of an antibody. 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 an antibody or TCR of interest. In an even more preferred aspect, the oligonucleotide encodes all or part of one or more CDRs or TCRs.

[0107] Probes based on the desired sequence of nucleic acids can be used to detect nucleic acids or similar nucleic acids, such as transcripts encoding a polypeptide of interest. The probe can include a labeling group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells that express the polypeptide.

[0108] IV. Polypeptide Expression In some aspects, there are polypeptide or nucleic acid molecules encoding a polypeptide of the present disclosure (e.g., TCR genes). These can be isolated by methods known in the art, e.g., from immune and isolated mouse B cells, and can be phage display expressed in any suitable recombinant expression system and assembled to form antibody molecules or can be produced by recombinant methods.

[0109] A. Expression Nucleic acid molecules can be used to express large amounts of polypeptide. When 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.

[0110] B. Vector In some aspects, expression vectors containing nucleic acid molecules encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains) are contemplated. Expression vectors containing nucleic acid molecules can 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 can encode fusion proteins, modified antibodies, antibody fragments, and their probes. In addition to control sequences that regulate transcription and translation, vectors and expression vectors can include nucleic acid sequences that perform other functions.

[0111] To express the polypeptides or peptides of the present disclosure, DNA encoding the polypeptide or peptide is inserted into an expression vector such that the gene region is operably linked to transcriptional and translational control sequences. In some aspects, a vector encoding a functionally complete human CH or CL immunoglobulin or TCR sequence with appropriate restriction sites engineered such that any variable region sequences can be readily inserted and expressed. In some aspects, a vector encoding a functionally complete human TCRα or TCRβ sequence with appropriate restriction sites engineered such that any variable sequence or CDR1, CDR2, and / or CDR3 can be readily inserted and expressed. Typically, an expression vector used in any host cell contains sequences for the maintenance of a plasmid or virus, as well as sequences for the cloning and expression of an exogenous nucleotide sequence. 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 inserting a nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using them are well known in the art.

[0112] C. Expression System There are numerous expression systems that contain at least a part or all of the above-described expression vector. These can be utilized for using prokaryotic and / or eukaryotic-based systems 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, systems of bacteria, mammals, yeast, and insect cells. 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 accurate modification and processing of the expressed foreign protein. Those skilled in the art can use an appropriate expression system to express the vector to produce a nucleic acid sequence or its cognate polypeptide, protein or peptide.

[0113] V. Methods of Gene Transfer Methods suitable for nucleic acid delivery to effect expression of a composition are contemplated to include substantially any method by which a nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into a cell, tissue or organism, as described herein or as known to those of skill in the art. Such methods include 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 incorporated herein by reference), including microinjection (Harland and Weintraub, 1985, 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 use of DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonoporation (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 particle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., each incorporated herein by reference), 1990; by U.S. Patent Nos. 5,302,523 and 5,464,765; by transformation via Agrobacterium (U.S. Patent Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); including, but not limited to, direct delivery of DNA such as by DNA uptake via drying / inhibition (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0114] A. Host Cell In another aspect, the use of host cells into which a recombinant expression vector has been introduced is contemplated. Antibodies can be expressed in a variety of cell types. Expression constructs encoding the antibodies can be transfected into cells by a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors can utilize control sequences that allow for replication and / or expression in both prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under the control of a promoter associated with T cell activation, such as one that is controlled by NFAT-1 or NF-κB, which are transcription factors that can both be activated upon T cell activation. By controlling antibody expression, T cells, such as tumor-targeting T cells, can sense their surroundings and implement real-time regulation of cytokine signaling in both the T cells themselves and the surrounding endogenous immune cells. Those skilled in the art will understand the conditions for incubating and maintaining the host cells to allow for replication of the vectors. Also understood and known are the techniques and conditions that allow for large-scale production of the vectors, as well as the production of the nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides.

[0115] For the stable transfection of mammalian cells, it is known that only a small fraction of the cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a selectable marker (e.g., for resistance to an antibiotic) is generally introduced into the host cell together with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified, inter alia, by drug selection (e.g., cells incorporating the selectable marker gene survive while other cells die) among other methods known in the art.

[0116] B. Isolation Nucleic acid molecules encoding the whole or one or both of the variable regions of the heavy, light, alpha, and beta chains of an antibody or TCR can be obtained from any source that produces the antibody. Methods for isolating mRNA encoding an antibody are well known in the art. See, e.g., Sambrook et al., supra. The sequences of the human heavy and light chain constant region genes are also known in the art. See, e.g., Kabat et al., 1991, supra. Next, nucleic acid molecules encoding the full-length heavy and / or light chains can be expressed in cells into which they are introduced, and the antibody can be isolated.

[0117] VI. Further Therapies A. Immunotherapy In some aspects, the method includes or excludes administering further therapy. In some aspects, the further therapy may include or exclude cancer immunotherapy. Cancer immunotherapy (also called immuno - oncology and abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be classified as active, passive, or hybrid (active and passive). These approaches utilize the fact that cancer cells often have on their surface molecules that can be detected by the immune system, known as tumor - associated antigens (TAAs); these are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy instructs 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. Immunotherapy is known in the art, and some are described below.

[0118] 1. Checkpoint inhibitors and combination treatments Aspects of the present disclosure can include or exclude the administration of immune checkpoint inhibitors, which are further described below.

[0119] 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 up - regulate PD - 1 and continue to express PD - 1 in peripheral tissues. Cytokines such as IFN - gamma induce the expression of PDL1 in epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD - 1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to tissues during an immune response. The inhibitors of the present disclosure can block one or more functions of PD - 1 and / or PDL1 activity.

[0120] 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.

[0121] In some aspects, a 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, a 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, a 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.

[0122] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PDL1 or PDL2 or a PD-1 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some aspects, the PDL1 inhibitor includes 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 is also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475 and is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab is also known as CT-011, hBAT, or hBAT-1 and is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 is also known as B7-DCIg and is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Further PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0123] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0124] 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 with the aforementioned antibodies in binding to an epitope on PD-1, PDL1, or PDL2 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 above-described antibody.

[0125] 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 an inhibitory signal to T cells. CTLA4 is similar to the T cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. When T cells are activated via the T cell receptor and CD28, the expression of CTLA-4, an inhibitory receptor for B7 molecules, increases. 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 inhibitor blocks the CTLA-4 and B7-1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0126] 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.

[0127] An anti-human - CTLA-4 antibody (or a VH domain and / or VL domain derived therefrom) suitable for use in the method of the present invention can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art 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; previously also known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the methods disclosed herein. The disclosure of each of the foregoing publications is incorporated herein by reference. For binding to CTLA-4, antibodies that compete with any of these antibodies recognized in the art can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0128] Further anti-CTLA-4 antibodies useful as checkpoint inhibitors in the methods and compositions of the present disclosure are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0129] 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 with the antibody described above in binding to an epitope on PD-1, B7-1, or B7-2, and / or binds to an epitope on PD-1, B7-1, or B7-2 that is the same as that of the antibody described above. 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 antibody described above.

[0130] 2. Inhibition of Costimulatory Molecules In some aspects, the immunotherapy comprises or excludes an inhibitor of a costimulatory molecule. 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. The inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0131] 3. Dendritic Cell Therapy Additional therapies may include or exclude dendritic cell therapy. Dendritic cell therapy elicits an anti-tumor response by causing dendritic cells to present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating the lymphocytes to kill other cells presenting the antigen. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells assist in targeting cancer antigens. An example of dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0132] One way to induce dendritic cells to present tumor antigens is by vaccinating with autologous tumor lysates or short peptides (small portions of proteins corresponding to protein antigens on cancer cells). These peptides are often given in combination with an adjuvant (a highly immunogenic substance) to enhance the immune and anti-tumor responses. Other adjuvants include proteins or other chemical substances, such as granulocyte macrophage colony-stimulating factor (GM-CSF), that attract and / or activate dendritic cells.

[0133] Dendritic cells can also be activated in vivo by causing tumor cells to express GM-CSF. This can be achieved by genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with a tumor-lytic virus that expresses GM-CSF.

[0134] Another strategy is to remove dendritic cells from a patient's blood and activate them ex vivo. Dendritic cells are activated in the presence of tumor antigens, which can be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of disrupted tumor cells). These cells (with a selective adjuvant) are then injected to elicit an immune response.

[0135] 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, which can induce dendritic cells to mature and provide immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 are used as antibody targets.

[0136] 4. CAR-T Cell Therapy Additional therapies may or may not include chimeric antigen receptor T cell therapy. A chimeric antigen receptor (CAR), also known as a chimeric immune receptor, chimeric T cell receptor or artificial T cell receptor, is an engineered receptor that combines an immune cell targeting cancer cells with a new specificity. Usually, these receptors transplant the specificity of monoclonal antibodies into T cells. The receptors are called chimeric because parts from different sources are fused. CAR-T cell therapy refers to a treatment using such transformed cells for cancer treatment.

[0137] The basic principle of CAR-T cell design includes a recombinant receptor that combines an antigen-binding function and a T cell activation function. A general premise of CAR-T cells is to artificially create T cells that target markers found on cancer cells. Scientists can remove T cells from a person, genetically modify them, and return them to the patient to attack cancer cells. When T cells are engineered to become CAR-T cells, they act as a "living drug". CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is a way to ensure that only cancerous tumor cells, not normal cells, are targeted. The specificity of CAR-T cells is determined by the selection of the molecule to be targeted.

[0138] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta). In some aspects, CAR-T therapy targets CD19.

[0139] 5. Cytokine Therapy Additional therapies may or may not include cytokine therapy. Cytokines are proteins produced by many types of cells present within tumors. They can regulate the immune response. Tumors often utilize cytokines to grow tumors and reduce the immune response. Due to these immunomodulatory effects, it becomes possible to use them as drugs to induce an immune response. Two commonly used cytokines are interferon and interleukin.

[0140] Interferons are produced by the immune system. They are usually involved in the antiviral response but are also used in cancer. They are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ), and type III (IFNλ).

[0141] Interleukins have numerous immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0142] 6. Adoptive T cell therapy Additional therapies may or may not include adoptive T cell therapy. Adoptive T cell therapy is a form of passive immunity by the transfusion of T cells (adoptive cell transfer). T cells are found in the blood and tissues and usually become activated when they encounter foreign pathogens. Specifically, T cells become activated when the surface receptors on T cells encounter cells that present a portion of a foreign protein on the surface antigen. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal tissues as well as 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. These cells can attack tumors, but the environment within the tumor is highly immunosuppressive and prevents immune-mediated tumor death.

[0143] Multiple methods have been developed for producing and obtaining T cells targeting tumors. T cells specific to tumor antigens can be removed from tumor samples (TIL) or filtered from the blood. Subsequent activation and culturing are carried out ex vivo and, as a result, re-injected. Activation can be performed through gene therapy or by exposing the T cells to tumor antigens.

[0144] B. Chemotherapy Additional therapies may or may not include chemotherapy. In some aspects, additional therapies include chemotherapy. Suitable classes of chemotherapeutic agents include (a) alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dacarbazine), (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) 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), methylhydrazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0145] 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, for example. Cisplatin is not absorbed orally and therefore must be delivered via other routes such as intravenous, subcutaneous, intratumoral or intraperitoneal injection, for example. Cisplatin can be used alone or in combination with other agents, and in certain settings an effective dose is contemplated for clinical use including from about 15 mg / m2 to about 20 mg / m2 for 5 days every 3 weeks for a total of 3 cycles. In some settings, the amount of cisplatin delivered to cells and / or a subject in combination 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.

[0146] Other suitable chemotherapeutic agents include antimicrotubule agents such as paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter / TNFα construct delivered via an adenovirus vector and doxorubicin has been shown to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0147] Doxorubicin has poor absorption and is preferably administered intravenously. In certain settings, appropriate intravenous doses for adults include from about 60 mg / m2 to about 75 mg / m2 at about 21-day intervals or from about 25 mg / m2 to about 30 mg / m2 for 2 or 3 consecutive days repeated at about 3- to 4-week intervals or about 20 mg / m2 once a week. The lowest doses should be used in the elderly in the presence of prior myelosuppression or neoplastic bone marrow infiltration caused by prior chemotherapy or when the drug is combined with other myelosuppressive agents.

[0148] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustard can include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson, and NEOSTAR® is available from Adria), another suitable chemotherapeutic agent. Suitable oral dosages for adults can include, for example, from about 1 mg / kg / day to about 5 mg / kg / day, and intravenous dosages can include, for example, initially from about 40 mg / kg to about 50 mg / kg in divided doses over about 2 days to about 5 days, or from about 10 mg / kg to about 15 mg / kg every about 7 days to about 10 days or twice a week, from about 3 mg / kg to about 5 mg / kg, or from about 1.5 mg / kg / day to about 3 mg / kg / day. Due to gastrointestinal side effects, the intravenous route is preferred. The drug can also be administered intramuscularly, by infiltration, or into a body cavity.

[0149] Further suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorode-oxyuridine; FudR). 5-FU can be administered to a subject at a dosage anywhere from about 7.5 to about 1000 mg / m2. Further, 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 the present disclosure pertains.

[0150] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), another suitable chemotherapeutic agent, is recommended for the treatment of advanced and metastatic pancreatic cancer and, therefore, would similarly be useful in the present disclosure for these cancers.

[0151] The amount of chemotherapeutic agent delivered to a patient can be variable. In one suitable aspect, the chemotherapeutic agent can be administered in an amount effective to cause arrest or regression of cancer in a host when chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent can be administered in an amount somewhere 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 for determination of an effective dosage. For example, such compounds can be tested in suitable animal model systems including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, etc. prior to testing in humans. As described in the examples, in vitro testing can also be used to determine suitable combinations and dosages.

[0152] C. Radiation Therapy Additional therapy may or may not include radiation therapy. In some aspects, additional or previous therapy includes radiation, such as ionizing radiation. As used herein, "ionizing radiation" means radiation that includes particles or photons that have sufficient energy or that can produce sufficient energy through nuclear interactions that produce ionization (acquisition or loss of electrons). Exemplary and preferred ionizing radiation is x-rays. Means for delivering x-rays to a target tissue or cell are well known in the art.

[0153] D. Surgery In some situations, further therapy includes or excludes surgery. Approximately 60% of people with cancer undergo some type of surgery, including prophylactic surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes excisions 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 the treatments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies of aspects of the present invention. Tumorectomy refers to the physical removal of at least part of a tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery, and microscopically-controlled surgery (Mohs surgery).

[0154] Removing part or all of the cancer cells, tissue, or tumor may create a cavity in the body. The treatment may be performed by perfusing, directly injecting, or topically applying additional anti-cancer therapy to the area. Such treatments may be repeated, for example, daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days, or every week, every 2 weeks, every 3 weeks, every 4 weeks, and every 5 weeks, or every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months (or any range derivable therein). These treatments may also be at various dosages.

[0155] 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, in isolated test samples, or by the methods described herein.

[0156] As used herein, the term "label" is intended to mean a directly or indirectly detectable compound or composition that, when conjugated directly or indirectly to a composition to be detected, such as a polynucleotide or a protein such as an antibody, results in a "labeled" composition. The term also includes sequences conjugated to polynucleotides that provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP). A label may be directly detectable itself (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, may catalyze a chemical change in a detectable substrate compound or composition. A label may be suitable for detection on a small scale or may be more suitable for high-throughput screening. Thus, suitable labels include, without limitation, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins including enzymes. A label may be simply detected or may be quantified. A response that is simply detected generally includes a response that merely confirms its presence, while a response that is quantified generally includes a response having a quantifiable (e.g., numerically reportable) value such as intensity, polarization, and / or other characteristics. In luminescence or fluorescence assay methods, a detectable response may occur directly using a luminophore or fluorophore associated with an assay component that is actually involved in the binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0157] Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally includes a change in or the occurrence of a luminescent signal. Suitable methods and luminophores for luminously labeling assay components are known in the art, e.g., Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 thIt is described in (ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0158] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methylcoumarin, pyrene, malachite 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.).

[0159] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component, such as a cell surface marker, present within or on the surface of a cell or tissue. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimide, succinimidyl esters, and halogenated sulfonyls, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group of the fluorescent label will depend on the binding site with either a linker, agent, marker, or second labeling agent.

[0160] Attachment of the fluorescent label may be direct to the cellular component or compound or, alternatively, can be 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.

[0161] Coupling a polypeptide with a low molecular weight hapten can enhance the sensitivity of antibodies in an assay method. Subsequently, the hapten can be specifically detected by a second reaction. For example, it is common to use haptens such as biotin that reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein that can react with a specific anti-hapten polypeptide. See Harlow and Lane (1988).

[0162] The agent to be conjugated can be linked directly or indirectly to the polypeptide using any of a number of available methods. For example, the agent can be attached to the hinge region of the reduced antibody component via a cross-linking agent such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP) through disulfide bond formation, or via the carbohydrate moiety in the Fc region of the antibody (Yu et al., 1994; Upeslacis et al., 1995; Price, 1995).

[0163] 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).

[0164] The polypeptide or its antigen-binding region of the present disclosure can be linked to another functional molecule such as a ligand, a cytotoxic molecule, a chemotherapeutic agent, or other agent described as a further therapeutic substance.

[0165] VIII. Formulation and Cultivation of Cells The medium in a particular situation can be prepared using as a basal medium a medium used for culturing animal cells, such as any one of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, modified MEM zinc option, IMDM, Medium 199, Eagle's MEM, αMEM, DMEM, Ham's, RPMI-1640, and Fisher's medium, as well as any combination thereof. However, the medium cannot be particularly limited thereto as long as it can be used for culturing animal cells. In particular, the medium can be serum-free or chemically defined.

[0166] The medium can be a serum-containing medium, a serum-free medium, or a xenogeneic-free medium. From the aspect of preventing contamination by components derived from heterologous animals, the serum can be derived from the same animal as that of the stem cells. A serum-free medium refers to a medium that does not have raw or unpurified serum, and thus can include a medium having purified blood-derived components or animal tissue-derived components (such as growth factors).

[0167] The medium may or may not contain any substitute for serum. Substitutes for serum may include materials appropriately containing albumin (such as albumin substitutes rich in lipids, 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 their equivalents. The substitute for serum can be prepared, for example, by the method disclosed in International Publication No. 98 / 30679 (which is incorporated herein by reference in its entirety). Alternatively, for greater convenience, any commercially available material can be used. Commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0168] In certain aspects, the medium may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 of the following other components: 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; corticosteron; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triiodo-I-thyronine). In certain aspects, one or more of these may be explicitly excluded.

[0169] In some embodiments, the medium further contains vitamins. In some embodiments, the medium contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (and any range derivable therefrom) of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. In some embodiments, the medium contains 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 embodiments, the vitamins contain or consist essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or combinations thereof or salts thereof. In some embodiments, the medium further contains protein. In some embodiments, the protein includes albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further contains one or more of corticosteron, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-L-thyronine, or combinations thereof. In some embodiments, the medium contains one or more of B-27® supplement, serum-free B-27® supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium contains, or further contains, amino acids, monosaccharides, and inorganic ions.In some aspects, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some aspects, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further comprises one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises one or more of the vitamins discussed herein, and / or one or more of the proteins discussed herein, and / or the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-L-thyronine, B-27® supplement, serum-free B-27® supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or consists essentially of these. In certain aspects, one or more of these may be explicitly excluded.

[0170] The medium can also contain one or more externally 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. In certain aspects, one or more of these may be explicitly excluded.

[0171] One or more of the culture components may be added at 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, 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 ng / L, ng / ml, μg / ml, mg / ml or any range derivable therein, or at a concentration of 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.

[0172] In certain aspects, the cells of the present disclosure are specifically 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 topical administration. In some cases, the cells are formulated for storage prior to use, and the cell preparation may contain one or more cryopreservatives such as DMSO (e.g., in 5% DMSO). The cell preparation may contain albumin, including human albumin, and certain preparations contain 2.5% human albumin. The cells may be specifically formulated for intravenous administration; for example, they are formulated for intravenous administration over less than 1 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 from the time of thawing.

[0173] In certain aspects, the cells of the present disclosure contain an exogenous TCR, which can be of defined antigen specificity. In some aspects, the TCR can be selected based on the absence or reduction of alloreactivity to an intended recipient (examples include a particular virus-specific TCR, a heterologous-specific TCR, or a cancer testis antigen-specific TCR). In examples 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 the expression of only the non-alloreactive exogenous TCR and thus giving rise to T cells that are non-alloreactive. In some aspects, the selection of the exogenous TCR may not necessarily be defined based on the absence of alloreactivity. In some aspects, the endogenous TCR gene has been modified by genome editing so as not to express a protein. Methods of gene editing, such as methods using the CRISPR / Cas9 system, are known in the art and are described herein.

[0174] 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 a CAR 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), the EGFR family including EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), Her2, IL-13Rα2, lambda (Lambda), Lewis-Y, kappa (Kappa), KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEM, cancer fetal antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV 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 a VEGF receptor (e.g., VEGFR2). The CAR may be a first-generation, second-generation, third-generation, or greater than third-generation CAR. The CAR may be bispecific for any two non-identical antigens, or specific for three or more non-identical antigens.

[0175] IX. Administration of Therapeutic Compositions The therapies provided herein may include the administration of combinations of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered by any suitable method known in the art. For example, the first and second cancer treatments may 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 in the same composition.

[0176] In certain aspects, the cells of the disclosure may be specially formulated and / or they may be cultured in a specific medium. The cells may be formulated in a manner suitable for delivery to a recipient without adverse effects.

[0177] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. Different therapies may be administered in one composition or in two or more compositions, such as two compositions, three compositions or four compositions. Various combinations of agents may be utilized.

[0178] The therapeutic compositions of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, locally, orally, transdermally, intraperitoneally, intraorbitally, by transplantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, locally, orally, transdermally, intraperitoneally, intraorbitally, by transplantation, by inhalation, intrathecally, intraventricularly, or intranasally. Appropriate dosages may be determined based on the type of disease being treated, the severity and course of the disease, the clinical condition of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0179] The treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route and formulation, are within the scope of the practitioner's decision-making skills in the clinical field. The unit dose does not necessarily have to be administered as a single injection, but may include continuous infusion over a set period. In some aspects, the unit dose includes a single administrable dose.

[0180] The exact amount of the therapeutic composition also depends on the judgment of the practitioner and is specific to each individual. Factors affecting the dose include the patient's physical and clinical condition, the route of administration, the intended treatment goal (symptom relief vs. cure), and the efficacy, stability and toxicity of the specific therapeutic substance or other therapies the subject may be receiving.

[0181] Cancers suitable for treatment include, but are not limited to, tumors of all types, locations, sizes, and characteristics. In some aspects, the cancer includes solid tumors. In some aspects, the method relates to reducing tumor volume or treating cancers that are recurrent and / or metastatic. 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 cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma of the cerebellum or cerebrum in children, 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, glioma of the visual pathway and hypothalamus, breast cancer, lymphatic cancer, bronchial adenoma / carcinoid, tracheal cancer, lung cancer, Burkitt lymphoma, carcinoid tumor, pediatric carcinoid tumor, gastrointestinal cancer of unknown primary origin, central nervous system lymphoma, primary cerebellar astrocytoma, pediatric cerebral astrocytoma / malignant glioma, pediatric cervical cancer, pediatric cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's, pediatric 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 tumor: extracranial, extragonadal or ovarian, gestational trophoblastic tumor, glioma of the brainstem, glioma, astrocytoma of the pediatric cerebrum, glioma of the pediatric visual pathway and hypothalamus, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, glioma of the hypothalamus and visual pathway, intraocular melanoma of the pediatric eye, islet cell cancer (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemia, acute lymphoblastic (also known as acute lymphocytic leukemia) leukemia, acute myeloid (also known as acute myelogenous leukemia) leukemia, chronic lymphocytic (also known as chronic lymphocytic leukemia) leukemia, chronic myelogenous (also known as chronic myeloid leukemia) 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 (old classification for all lymphomas other than Hodgkin) lymphoma, primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of the bone, medulloblastoma of the pediatric brain, intraocular (eye) melanoma, Merkel cell cancer, malignant mesothelioma of the adult, mesothelioma of the pediatric, metastatic cervical squamousis suitable for treating neck cancer, mouth cancer, multiple endocrine neoplasia, multiple myeloma / plasma cell tumor, fungating polyps, myelodysplastic syndrome, myelodysplastic / myeloproliferative disease, chronic myelogenous leukemia, adult acute myelogenous leukemia, pediatric acute myelogenous leukemia, multiple myeloma, chronic myeloproliferative disorder, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial / stromal tumor), ovarian germ cell tumor, low malignant potential ovarian tumor, pancreatic cancer, pancreatic islet cell paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineoblastoma, pinealoblastoma and supratentorial primitive neuroectodermal tumor, pediatric pituitary adenoma, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, pediatric salivary gland carcinoma sarcoma, Ewing sarcoma family tumor, Kaposi sarcoma, soft tissue sarcoma, uterine cesarean syndrome sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell cancer, squamous cell cancer of the neck of unknown primary origin, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, pediatric T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma, pediatric thymoma, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, glioma of the visual pathway and hypothalamus, pediatric vulvar cancer, as well as Wilms tumor (kidney cancer).

[0182] X. Kit Certain aspects of the present invention also relate to kits comprising the compositions of the present disclosure or compositions for practicing the methods of the present invention. In some aspects, the kits can be used to evaluate one or more biomarkers or HLA types. In certain aspects, the kit comprises 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 therefrom, or comprises 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, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therefrom, or alternatively comprises 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 therefrom.

[0183] The kit can include components that can be individually packaged or disposed within a container, such as tubes, bottles, vials, syringes, or other suitable container means.

[0184] Individual components may be provided in the kit in concentrated amounts; in some aspects, the components are provided individually at the same concentration as they are in solution with other components. The concentration of the components can be provided as 1-fold, 2-fold, 5-fold, 10-fold or 20-fold or more.

[0185] In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. Further, the kit can include a sample that is a negative or positive control for the methylation of one or more biomarkers.

[0186] It is contemplated that any method or composition described herein can be practiced with respect to any other method or composition described herein, and that different aspects can be combined. The original claims are intended to cover claims that depend compoundingly on any of the filed claims or combinations of filed claims.

Examples

[0187] XI. Examples The following examples are included to demonstrate preferred embodiments of the disclosure. It will be recognized by those skilled in the art that the techniques disclosed in the following examples are those that the inventors have found to function well in the practice of the disclosure, and thus represent preferred modes for its practice. However, those skilled in the art will recognize that, in light of the disclosure, many modifications can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0188] Example 1: Generation of TCR gene-modified T cells having this TCR sequence for adoptive T cell therapy for the treatment of HLA-A11 and VCY-expressing cancer patients. The inventors of the present invention identified the HLA-A11-restricted epitope of human CT antigen VCY (amino acid sequence: SSQPSPSGPK - SEQ ID NO:15) by mass spectrometry (Figs. 1-2). However, there are no reports on the generation of specific T cells, the identification of the correlated T cell receptor (TCR), and the development of TCR-engineered T cells (TCR-T). Using this peptide and the correlated tetramer (Fig. 3), the inventors of the present invention successfully generated in vitro an antigen-specific CTL cell line that was demonstrated to kill tumor cells presenting this peptide endogenously (Figs. 4-6). Based on this finding, the inventors of the present invention cloned the TCR sequence (including the TCR α-chain and β-chain) from the CTL cell line. Next, the inventors of the present invention constructed a retroviral vector containing the full-length TCR α-chain and β-chain together with a linker capable of equally expressing both chains. Using this retroviral vector, the inventors of the present invention introduced this TCR into allogeneic peripheral blood mononuclear cells (PBMCs) and successfully generated a specific TCR-T cell line (Fig. 7). This TCR-T cell line was able to recognize its cognate epitope and kill tumor cells expressing the VCY antigen, but the TCR-T cells did not kill the negative control target (Figs. 8-9). Based on these results, the inventors of the present invention plan to evaluate the safety and efficacy of this TCR-T cell therapy for the treatment of HLA-A*1101-positive patients with advanced or recurrent cancer in a non-randomized dose-escalation Phase I / II trial. Based on the experiments conducted so far, the inventors of the present invention generate and expand TCR-T cells using autologous PBMCs. After functional detection (phenotype, killing ability, etc.), the TCR-T cells are injected into patients in a stepwise dosing strategy to evaluate safety. In the absence of dose-limiting toxicity (DLT), the inventors of the present invention proceed to determine the antitumor efficacy by disease stage diagnosis evaluation and describe it as complete response (CR), partial response (PR), or progressive disease (PD) (RECIST criteria). The inventors of the present invention also determine the duration of persistence and progression-free survival of the transplanted TCR-T.

[0189] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present invention. Although the compositions and methods of the present invention have been described in 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 the order of the steps of the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it is apparent that certain agents chemically and physiologically related can be used in place of the agents described herein, and at the same time, the same or similar results can be obtained. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the scope of the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

**Claim 1** A polypeptide comprising a single-chain T cell receptor (TCR) comprising a TCR-α polypeptide and a TCR-β polypeptide, wherein the TCR-α polypeptide comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and the TCR-β polypeptide comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively, a polypeptide. **Claim 2** A polypeptide comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

8. **Claim 3** The polypeptide according to claim 2, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:

8. **Claim 4** The polypeptide according to claim 2 or 3, wherein the variable region comprises CDR1, CDR2, and / or CDR3. **Claim 5** The polypeptide according to claim 4, wherein the variable region comprises a CDR1 having at least 80% sequence identity to SEQ ID NO:

6. **Claim 6** The polypeptide according to claim 4 or 5, wherein the variable region comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:

7. **Claim 7** The polypeptide according to claim 5 or 6, wherein the variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 and / or a CDR2 comprising the amino acid sequence of SEQ ID NO:

7. **Claim 8** The polypeptide according to any one of claims 2 to 7, wherein the variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

4. **Claim 9** The polypeptide according to claim 8, wherein the variable region comprises the amino acid sequence of SEQ ID NO:

4. **Claim 10** The polypeptide according to any one of claims 2 to 9, comprising a T cell receptor alpha (TCR-α) variable region. **Claim 11** The polypeptide according to claim 10, comprising a TCR-α variable region and a constant region. **Claim 12** The polypeptide according to any one of claims 2 to 11, further comprising a signal peptide. **Claim 13** The polypeptide according to claim 12, wherein the signal peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:

5. **Claim 14** The polypeptide according to claim 13, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

5.

15. A polypeptide comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

14.

16. The polypeptide according to claim 15, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:

14.

17. The polypeptide according to claim 15 or 16, wherein the variable region comprises CDR1, CDR2, and / or CDR3.

18. The polypeptide according to claim 17, wherein the variable region comprises a CDR1 having at least 80% sequence identity to SEQ ID NO:

12.

19. The polypeptide according to claim 17 or 18, wherein the variable region comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:

13.

20. The polypeptide according to claim 18 or 19, wherein the variable region comprises 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.

21. The polypeptide according to any one of claims 15 to 20, wherein the variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

10.

22. The polypeptide according to claim 21, wherein the variable region comprises the amino acid sequence of SEQ ID NO:

10.

23. The polypeptide according to any one of claims 15 to 22, comprising a T cell receptor beta (TCR-b) variable region.

24. The polypeptide according to claim 23, comprising a TCR-b variable region and a constant region.

25. The polypeptide according to any one of claims 15 to 24, further comprising a signal peptide.

26. The polypeptide according to claim 25, wherein the signal peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:

11.

27. The polypeptide according to claim 26, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

11.

28. An engineered T cell receptor (TCR) comprising a TCR-a polypeptide and a TCR-b polypeptide, The TCR-α polypeptide comprises a CDR3 that includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO:8, and the TCR-β polypeptide comprises a CDR3 that includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO:14, the engineered TCR. **Claim 29** The TCR according to claim 28, wherein the TCR-α polypeptide comprises a CDR3 that includes the amino acid sequence of SEQ ID NO:8, and the TCR-β polypeptide comprises a CDR3 that includes the amino acid sequence of SEQ ID NO:

14. **Claim 30** The TCR according to claim 28 or 29, comprising a TCR-α polypeptide that includes a variable region comprising CDR1, CDR2, and CDR3, and a TCR-β polypeptide that includes a variable region comprising CDR1, CDR2, and CDR3. **Claim 31** The TCR according to claim 30, wherein the TCR-α polypeptide comprises a CDR1 having at least 80% sequence identity to SEQ ID NO:6 and / or the TCR-β polypeptide comprises a CDR1 having at least 80% sequence identity to SEQ ID NO:

12. **Claim 32** The TCR according to claim 31, wherein the TCR-α polypeptide comprises a CDR1 that includes the amino acid sequence of SEQ ID NO:6, and the TCR-β polypeptide comprises a CDR1 that includes the amino acid sequence of SEQ ID NO:

12. **Claim 33** The TCR according to any one of claims 30 to 32, wherein the TCR-α polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:7, and the TCR-β polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:

13. **Claim 34** The TCR according to claim 33, wherein the TCR-α polypeptide comprises a CDR2 that includes the amino acid sequence of SEQ ID NO:7, and the TCR-β polypeptide comprises a CDR2 that includes the amino acid sequence of SEQ ID NO:

13. **Claim 35** The TCR according to any one of claims 30 to 34, wherein the CDR1, CDR2, and CDR3 of the TCR-α polypeptide each include the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and the CDR1, CDR3, and CDR3 of the TCR-β polypeptide each include the amino acid sequences of SEQ ID NOs:12, 13, and 14, respectively.

36. The TCR, according to any one of claims 30 to 35, wherein the TCR-α polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, and the TCR-β polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

10.

37. The TCR according to claim 36, wherein the TCR-α polypeptide comprises the amino acid sequence of SEQ ID NO:4, and the TCR-β polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

10.

38. The TCR according to any one of claims 28 to 37, which comprises a modification or is chimeric.

39. The TCR according to any one of claims 28 to 38, wherein the TCR-α polypeptide and the TCR-β polypeptide are functionally linked.

40. The TCR according to claim 39, wherein the TCR-α polypeptide and the TCR-β polypeptide are functionally linked via a peptide bond.

41. The TCR according to claim 39, which is a single-chain TCR.

42. The TCR according to claim 40 or 42, wherein the TCR-α polypeptide and the TCR-β polypeptide are on the same polypeptide, and TCR-β is amino-proximal to TCR-α.

43. The TCR according to claim 40 or 42, wherein the TCR-α polypeptide and the TCR-β polypeptide are on the same polypeptide, and TCR-α is amino-proximal to TCR-β.

44. The TCR according to any one of claims 40 to 43, which comprises a linker between the TCR-α polypeptide and the TCR-β polypeptide.

45. The TCR according to any one of claims 40 to 44, wherein the linker comprises glycine and serine residues.

46. A peptide having at least 66% sequence identity to SEQ ID NO:

15.

47. The peptide according to claim 46, which comprises SEQ ID NO:

15.

48. The peptide according to claim 46, which comprises at least 6 consecutive amino acids of SEQ ID NO:

15.

49. The peptide according to claim 46, which comprises at least 7 consecutive amino acids of SEQ ID NO:

15.

50. The peptide according to claim 46, which comprises at least 8 consecutive amino acids of SEQ ID NO:

15.

51. The peptide according to any one of claims 46 to 50, comprising at least 77% sequence identity to SEQ ID NO:

15.

52. The peptide according to any one of claims 46 to 51, comprising at least 88% sequence identity to SEQ ID NO:

15.

53. The peptide according to any one of claims 46 to 52, consisting of 7 amino acids.

54. The peptide according to any one of claims 46 to 52, consisting of 8 amino acids.

55. The peptide according to any one of claims 46 to 52, consisting of 9 amino acids.

56. The peptide according to claim 46, consisting of SEQ ID NO:

15.

57. The peptide according to any one of claims 46 to 56, which is immunogenic.

58. The peptide according to any one of claims 46 to 57, which is modified.

59. The peptide according to claim 58, wherein the modification comprises conjugation to a molecule.

60. The peptide according to claim 58 or 59, wherein the molecule comprises an antibody, a lipid, an adjuvant, or a detection moiety.

61. The peptide according to any one of claims 46 to 60, having 1, 2, or 3 substitutions with respect to the peptide of SEQ ID NO:

15.

62. A polypeptide comprising the peptide according to any one of claims 46 to 61.

63. A composition comprising at least one MHC polypeptide and the peptide or polypeptide according to any one of claims 46 to 62.

64. The composition according to claim 63, wherein the MHC polypeptide and / or the peptide is conjugated to a detection tag.

65. The composition according to claim 63 or 64, wherein the MHC polypeptide and the peptide are functionally linked.

66. The composition according to claim 65, wherein the MHC polypeptide and the peptide are functionally linked via a peptide bond.

67. The composition according to claim 65, wherein the MHC polypeptide and the peptide are functionally linked by van der Waals forces.

68. The composition according to any one of claims 63 to 67, wherein at least two MHC polypeptides are linked to one peptide.

69. The composition according to any one of claims 63 to 68, wherein the average ratio of MHC polypeptide to peptide is 4:

1.

70. A molecular complex comprising a peptide or polypeptide according to any one of claims 46 to 62 and an MHC polypeptide.

71. A peptide-specific binding molecule that specifically binds to a peptide or polypeptide according to any one of claims 46 to 62 or to the molecular complex according to claim 70.

72. The binding molecule according to claim 71, which is an antibody, a TCR-mimicking antibody, a scFv, a nanobody, an aptamer, or a DARPin.

73. A method for producing peptide-specific immune effector cells, comprising the following steps: (a) obtaining a starting population of immune effector cells, and (b) contacting the starting population of immune effector cells with a peptide or polypeptide according to any one of claims 46 to 62 or with the molecular complex according to claim 70, thereby generating peptide-specific immune effector cells.

74. The method according to claim 73, wherein the contacting step is further defined as co-culturing the starting population of immune effector cells with an antigen-presenting cell (APC), an artificial antigen-presenting cell (aAPC), or an artificial antigen-presenting surface (aAPS), wherein the APC, aAPC, or aAPS presents the peptide on its surface.

75. The method according to claim 74, wherein the APC is a dendritic cell.

76. The method according to any one of claims 73 to 75, wherein the immune effector cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, NKT cells.

77. The method according to any one of claims 73 to 76, wherein the immune effector cells are differentiated from mesenchymal stem cells (MSC) or induced pluripotent stem (iPS) cells.

78. T cells are CD8 + T cells, CD4 + 78. The method of claim 77, wherein the T cell is a γδ T cell.

79. The method according to claim 77, wherein the T cell is a cytotoxic T lymphocyte (CTL).

80. The method according to any one of claims 73 to 79, wherein the obtaining step comprises isolating a starting population of immune effector cells from peripheral blood mononuclear cells (PBMC).

81. The method according to any one of claims 73 to 80, wherein the starting population of immune effector cells is obtained from a subject.

82. The method according to claim 81, wherein the subject is human.

83. The method according to claim 81, wherein the subject has cancer.

84. The method according to claim 83, wherein the cancer comprises a VCY CT antigen-positive cancer.

85. The method according to claim 84, wherein the cancer comprises cancer cells that are positive for the peptide of SEQ ID NO:

15.

86. The method according to any one of claims 83 to 85, wherein the subject has been determined to have a VCY CT antigen-positive cancer.

87. The method according to claim 86, wherein the subject has been determined to have cancer cells that are positive for the peptide of SEQ ID NO:

15.

88. The method according to any one of claims 83 to 87, wherein the subject has been diagnosed with cancer.

89. The method according to any one of claims 73 to 88, further comprising introducing the peptide or the nucleic acid encoding the peptide into dendritic cells prior to co-culture.

90. The method according to claim 89, wherein the peptide or the nucleic acid encoding the peptide is introduced by electroporation.

91. The method according to claim 89, wherein the peptide or the nucleic acid encoding the peptide is introduced by adding the peptide or the nucleic acid encoding the peptide to a dendritic cell culture medium.

92. The method according to any one of claims 73 to 91, wherein the immune effector cells are co-cultured with a second population of dendritic cells into which the peptide or the nucleic acid encoding the peptide has been introduced.

93. The method according to any one of claims 73 to 92, wherein after co-culture, a population of CD8 or CD4-positive and peptide-MHC tetramer-positive T cells is purified from the immune effector cells.

94. The method according to claim 93, wherein a clonal population of peptide-specific immune effector cells is generated by limiting dilution or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

95. The method according to claim 94, further comprising cloning of the T cell receptor (TCR) from the clonal population of peptide-specific immune effector cells.

96. The method according to claim 95, wherein the cloning of the TCR is cloning of the TCRα and β chains.

97. The method according to claim 95 or claim 96, wherein the TCR is cloned using the 5'-cDNA end rapid amplification (RACE) method.

98. The method according to claim 97, wherein the cloned TCR is subcloned into an expression vector.

99. The method according to claim 98, wherein the expression vector is a retroviral vector or a lentiviral vector.

100. The method according to claim 98 or 99, further comprising the step of transducing a host cell with the expression vector to produce engineered cells expressing the TCR.

101. The method according to claim 100, wherein the host cell is an immune cell.

102. The method according to any one of claims 73 to 101, wherein the immune cell is a T cell and the engineered cell is an engineered T cell.

103. The T cell is CD8 + The method according to claim 102, wherein the T cell is a CD8 T cell, a CD4+ T cell, or a γδ T cell, and the engineered cell is an engineered T cell.

104. The method according to any one of claims 73 to 103, wherein the starting population of immune effector cells is obtained from a subject having cancer and the host cell is allogeneic or autologous to the subject.

105. The method according to any one of claims 100 to 104, wherein a population of engineered T cells that are CD8 or CD4 positive and peptide-MHC tetramer positive is purified from the transduced host cells.

106. The method according to any one of claims 73 to 105, wherein a clonal population of peptide-specific engineered T cells is generated by limiting dilution or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

107. A method for cloning a T cell receptor (TCR) comprising the following steps: (a) obtaining a starting population of immune effector cells; (b) contacting the starting population of immune effector cells with a peptide or polypeptide according to any one of claims 46 to 62, thereby generating peptide-specific immune effector cells; (c) purifying the immune effector cells specific for the peptide; (d) isolating the TCR sequence from the purified immune effector cells.

108. The method according to claim 107, wherein the contacting step is further defined as co-culturing the starting population of immune effector cells with an antigen-presenting cell (APC), wherein the APC presents the peptide on its surface.

109. The method according to claim 108, wherein the APC is a dendritic cell.

110. The method according to any one of claims 107 to 109, wherein the immune effector cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, or NKT cells.

111. The method according to any one of claims 107 to 110, wherein the immune effector cells are differentiated from mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells.

112. The T cell is CD8 + T cell, CD4 + The method according to claim 110 or 111, wherein the T cell is a CD8 T cell, a CD4 T cell, or a γδ T cell.

113. The method according to any one of claims 110 to 112, wherein the T cells are cytotoxic T lymphocytes (CTLs).

114. The method according to any one of claims 107 to 113, wherein the obtaining step comprises isolating a starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs).

115. The method according to any one of claims 107 to 114, wherein the starting population of immune effector cells is obtained from a subject.

116. The method according to claim 115, wherein the subject is human.

117. The method according to claim 115 or 116, wherein the subject has cancer.

118. The method according to claim 117, wherein the cancer comprises a VCY CT antigen-positive cancer.

119. The method according to claim 118, wherein the cancer comprises cancer cells that are positive for the peptide of SEQ ID NO:

15.

120. The method according to any one of claims 115 to 119, wherein the subject has been determined to have a VCY CT antigen-positive cancer.

121. The method according to claim 120, wherein the subject has been determined to have cancer cells that are positive for the peptide of SEQ ID NO:

15.

122. The method according to any one of claims 107 to 121, further comprising, prior to co-culture, introducing the peptide or the nucleic acid encoding the peptide into dendritic cells.

123. The method according to claim 122, wherein the peptide or the nucleic acid encoding the peptide is introduced by electroporation.

124. The method according to claim 122, wherein the peptide or the nucleic acid encoding the peptide is introduced by adding the peptide or the nucleic acid encoding the peptide to the culture medium of the dendritic cells.

125. The method according to any one of claims 109 to 124, wherein the immune effector cells are co-cultured with a second population of dendritic cells into which the peptide or the nucleic acid encoding the peptide has been introduced.

126. The method according to any one of claims 107 to 125, wherein the purification step is defined as purifying a population of CD8-positive and peptide-MHC tetramer-positive T cells from immune effector cells after co-culture.

127. The method according to claim 126, wherein the population of CD8-positive and peptide-MHC tetramer-positive T cells is purified by fluorescence-activated cell sorting (FACS).

128. The method according to claim 127, wherein the purification step further comprises generating a clonal population of peptide-specific immune effector cells by limiting dilution or serial dilution of the selected cells followed by expansion of individual clones by a rapid expansion protocol.

129. The method according to claim 128, further comprising cloning of the T cell receptor (TCR) from the clonal population of peptide-specific immune effector cells.

130. The method according to any one of claims 107 to 129, further comprising sequencing the TCRα and / or β genes and / or performing group-based lymphocyte interaction by paratope hot spot (GLIPH) analysis.

131. The method according to claim 129 or 130, wherein the cloning of the TCR is cloning of the TCRα and β chains.

132. The method according to claim 131, wherein the TCRα and β chains are cloned using the 5'-cDNA end rapid amplification (RACE) method.

133. The method according to claim 132, wherein the cloned TCR is subcloned into an expression vector.

134. The method according to claim 133, wherein the expression vector contains a linker domain between the TCRα sequence and the TCRβ sequence.

135. The method according to claim 134, wherein the linker domain contains a sequence encoding one or more peptide cleavage sites.

136. The method according to claim 135, wherein the one or more cleavage sites are furin cleavage sites and / or P2A cleavage sites.

137. The method according to claim 136, wherein the TCRα sequence and the TCRβ sequence are linked by an IRES sequence.

138. The method according to any one of claims 133 to 137, wherein the expression vector is a retroviral vector or a lentiviral vector.

139. The method according to claim 138, wherein the host cell is transduced with an expression vector to produce engineered cells expressing the TCR α-chain and β-chain.

140. The method according to claim 139, wherein the host cell is an immune cell.

141. Peptide-specific engineered T cells produced by the method according to any one of claims 73 to 140.

142. A TCR produced by the method according to any one of claims 107 to 138.

143. A fusion protein comprising a TCR according to any one of claims 28 to 45 or 142 and a CD3 binding region.

144. The fusion protein according to claim 46, 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.

145. The TCR according to any one of claims 28 to 45, or the fusion protein according to claim 143 or 144, wherein the TCR or fusion protein is conjugated to a detection agent or a therapeutic agent.

146. The TCR or fusion protein according to claim 145, wherein the agent comprises a fluorescent molecule, a radioactive molecule, or a toxin.

147. A nucleic acid encoding a polypeptide according to any one of claims 2 to 27 or 62, a TCR according to any one of claims 28 to 45, 142, 145, or 146, a peptide according to any one of claims 43 to 64, or a fusion protein according to any one of claims 143 to 146.

148. The nucleic acid according to claim 147, which is RNA.

149. The nucleic acid according to claim 147, which is DNA or cDNA encoding the peptide or polypeptide or a complement of the peptide or polypeptide.

150. The nucleic acid according to claim 147, having at least 70% sequence identity to one of SEQ ID NO: 1, 2, or a fragment thereof.

151. A nucleic acid expression vector comprising the nucleic acid according to any one of claims 147 to 150.

152. The vector according to claim 151, comprising a promoter that directs the expression of the nucleic acid.

153. The vector according to claim 152, wherein the promoter comprises a murine stem cell virus (MSCV) promoter.

154. The vector according to any one of claims 151 to 153, comprising TCR-a and TCR-b genes.

155. A cell comprising the polypeptide according to any one of claims 2 to 27 or 62, the TCR according to any one of claims 28 to 45, 142, 145, or 146, the fusion protein according to any one of claims 143 to 146, the nucleic acid according to any one of claims 147 to 150, or the vector according to any one of claims 151 to 154.

156. The cell according to claim 155, comprising a stem cell, a progenitor cell, an immune cell, or a natural killer (NK) cell.

157. The cell according to claim 156, comprising a hematopoietic stem cell or hematopoietic progenitor cell, a T cell, a cell differentiated from mesenchymal stem cells (MSCs), or an induced pluripotent stem cell (iPSC).

158. The cell according to claim 156 or 157, isolated from or derived from peripheral blood mononuclear cells (PBMCs).

159. T cells are cytotoxic T lymphocytes (CTLs), CD8 + T cells, CD4 + The cell according to claim 157 or 158, comprising invariant NK T (iNKT) cells, gamma-delta T cells, NKT cells, or regulatory T cells.

160. The cell according to any one of claims 155 to 159, isolated from a cancer patient.

161. An in vitro isolated dendritic cell comprising the peptide or polypeptide according to any one of claims 46 to 61 or 62, the nucleic acid according to any one of claims 147 to 150, or the vector according to any one of claims 151 to 154.

162. The dendritic cell according to claim 161, which is a mature dendritic cell.

163. The dendritic cell according to claim 161 or 162, wherein the cell has HLA-A, HLA-B, or HLA-C type.

164. The dendritic cell according to claim 163, wherein the cell is HLA-A or HLA-A*1101 type.

165. A composition comprising the polypeptide according to any one of claims 2 to 27 or 62, the TCR according to any one of claims 28 to 45, 142, 145, or 146, the fusion protein according to any one of claims 143 to 146, the nucleic acid according to any one of claims 147 to 150, the vector according to any one of claims 151 to 154, or the cell according to any one of claims 155 to 164.

166. The composition according to claim 165, formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection.

167. The composition according to claim 165 or 166, wherein the peptide is contained in liposomes, lipid-containing nanoparticles, or lipid-based carriers.

168. The composition according to any one of claims 165 to 167, which is formulated as a vaccine.

169. The composition according to any one of claims 165 to 168, further comprising an adjuvant.

170. The composition according to any one of claims 165 to 169, which has been confirmed to be serum-free, mycoplasma-free, endotoxin-free, and / or sterile.

171. A method for producing engineered cells, comprising introducing into cells the nucleic acid according to any one of claims 147 to 150 or the vector according to any one of claims 151 to 154.

172. The method according to claim 171, further comprising culturing the cells in a medium, incubating the cells under conditions that allow cell division, screening the cells, and / or freezing the cells.

173. A method for treating cancer in a subject, comprising administering to a subject in need thereof the composition according to any one of claims 165 to 170 or the cells according to any one of claims 141 or 155 to 160.

174. A method for treating or preventing cancer in a subject, comprising administering to a subject in need thereof the composition according to any one of claims 165 to 170 or the cells according to any one of claims 141 or 155 to 160.

175. The method according to claim 173 or 174, wherein the cancer is selected from non-small cell lung cancer and melanoma.

176. A method for stimulating an immune response in a subject, comprising administering to a subject in need thereof the composition according to any one of claims 165 to 170 or the cells according to any one of claims 141 or 155 to 160.

177. The method according to any one of claims 173 to 176, wherein the subject is a human subject.

178. The method according to any one of claims 173 to 177, wherein the cells are autologous.

179. The method according to any one of claims 173 to 177, wherein the cells are allogeneic.

180. The method according to any one of claims 173 to 179, wherein the subject has been previously treated for cancer.

181. The method according to claim 180, wherein the subject has been determined to be resistant to previous treatments. **Claim 182** The method according to any one of claims 173 to 181, further comprising administering a further therapy. **Claim 183** The method according to any one of claims 173 to 182, wherein the cancer comprises a cancer of stage I, II, III, or IV. **Claim 184** The method according to any one of claims 173 to 183, wherein the cancer comprises a metastatic and / or recurrent cancer. **Claim 185** The method according to any one of claims 173 to 184, wherein the cancer is a VCY CT antigen-positive cancer. **Claim 186** The method according to any one of claims 173 to 185, wherein the subject has been determined to have VCY CT antigen-positive cancer cells. **Claim 187** The method according to any one of claims 173 to 185, wherein the subject has been determined to have cancer cells expressing the peptide of SEQ ID NO:

15. **Claim 188** The method according to any one of claims 173 to 187, wherein the subject is HLA-A positive and / or has been determined to be HLA-A positive. **Claim 189** The method according to any one of claims 173 to 185, wherein the subject is HLA-A1101 positive and / or has been determined to be HLA-A1101 positive. **Claim 190** A method for predicting the prognosis of a patient or for detecting a T cell response in a patient, the method comprising contacting a biological sample from the patient with a peptide or polypeptide according to any one of claims 46 to 61 or a molecular complex according to claim 70. **Claim 191** The method according to claim 190, wherein the biological sample comprises a blood sample or a fraction thereof. **Claim 192** The method according to claim 191, wherein the biological sample comprises lymphocytes. **Claim 193** The method according to claim 192, wherein the biological sample comprises a fractionated sample comprising lymphocytes. **Claim 194** The method according to any one of claims 190 to 193, wherein the peptide is linked to a solid support. **Claim 195** The method according to claim 194, wherein the peptide is conjugated to a solid support or bound to an antibody conjugated to a solid support. **Claim 196** The method of claim 194, wherein the solid support comprises a microplate, beads, a glass surface, a slide, or a cell culture dish. **Claim 197** The method according to any one of claims 190 to 196, wherein detecting the T cell response comprises detecting binding of the peptide to the T cell or TCR. **Claim 198** A kit comprising a peptide or polypeptide according to any one of claims 46 to 62 in a container. **Claim 199** The kit of claim 198, wherein the peptide is included in a pharmaceutical preparation. **Claim 200** The kit of claim 199, wherein the pharmaceutical preparation is formulated for parenteral administration or inhalation. **Claim 201** The kit of claim 198, wherein the peptide is included in a cell culture medium.

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