Peptides and engineered T cell receptors targeting the NDC80 antigen and methods of use

Abstract

The present disclosure provides engineered T cell receptors (TCRs), cells that contain TCRs, and methods for making and using TCRs. The present disclosure relates to TCRs that specifically recognize epitopes derived from tumor antigen NDC80 CT. Thus, aspects of the present disclosure relate to engineered T cell receptors (TCRs), nucleic acids that code TCRs, and cells that contain the nucleic acids and TCRs. Also provided are compositions that include the cells, nucleic acids, or engineered TCRs of the present disclosure, methods for making the cells, and methods for using aspects of the present disclosure for therapeutic treatment. TIFF2024538169000023.tif71128

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 256,982, filed October 18, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing prepared in accordance with ST.26 format, which is incorporated herein by reference in its entirety. The sequence listing, created on October 11, 2022, is entitled MDACP1311WO and is 25,004 bytes in size.

[0003] I. FIELD OF THEINVENTION The present invention relates to the field of cancer therapy. [Background technology]

[0004] II. Background Adoptive T cell therapy is one of the potentially powerful cancer treatments in which natural T cells are genetically modified to make them tumor-specific and improve their ability to destroy tumor cells. Genetically modified T cells can express chimeric antigen receptors (CARs) or T cell receptors (TCRs) and have shown impressive results in multiple clinical trials. TCR-engineered T (TCR-T) cells show great promise against tumors. The efficacy of TCR depends on its interaction with peptide major histocompatibility complex (pMHC), a complex formed by peptides bound to MHC. Intracellular antigens are cleaved into peptide chains and presented by MHC molecules to form pMHC. Cytoplasmic proteins expressed by class I MHC proteins, most of which are defective ribosomal translation products, are 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 to class II MHC proteins T cell receptors from T cells, which must match the patient's human leukocyte antigen (HLA) alleles, and recognize these pMHCs, triggering 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). There is a need for engineering TCRs that are directed against cancer-specific antigens and are useful in the treatment of cancer. Summary of the Invention

[0005] The present disclosure provides compositions and methods, including peptides, engineered T cell receptors (TCRs), cells comprising the peptides and TCRs, and methods of making and using the peptides and TCRs. The present disclosure relates to TCRs that specifically recognize the NDC80 CT antigen, such as a peptide having the amino acid sequence of GLNEEIARV (SEQ ID NO:15).

[0006] Thus, the present disclosure: Also disclosed is a polypeptide comprising an antigen-binding variable region comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF2024538169000002.tif4128. It is a polypeptide comprising an antigen-binding variable region including a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF2024538169000003.tif4128.

[0007] The present disclosure also provides a TCR-a polypeptide comprising: and a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF2024538169000004.tif4128, Provided are T cell receptors (TCRs) and engineered TCRs, such as a T cell receptor (TCR) comprising a TCR-a polypeptide and a TCR-b polypeptide, comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF2024538169000005.tif4128. The TCR-a polypeptide comprises: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% for TIFF2024538169000006.tif4128 (or any range derivable therein), or 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, and the TCR-b 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% for TIFF2024538169000007.tif4128 (or any range derivable therein), or a CDR3 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. and the TCR-b polypeptide may comprise a CDR3 comprising the amino acid sequence of It may comprise a CDR3 comprising the amino acid sequence of TIFF2024538169000009.tif4128.

[0008] Also described 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 portion (Fab), a single chain variable fragment (scFv), a single domain antibody, or a single chain antibody. Exemplary CD3 specific fragment antigen binding portions (Fab) are known in the art. For example, US20180222981, which is incorporated herein by reference, discloses variable regions that specifically bind to CD3 and 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 also relates to peptides that comprise at least 66% sequence identity to the peptide of SEQ ID NO: 15. Also disclosed are polypeptides that comprise the peptides of the present disclosure. The peptides or polypeptides may comprise 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% sequence identity to SEQ ID NO: 15. (or any range derivable therein), 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] The present disclosure also relates to a molecular complex comprising the peptide or polypeptide of the present disclosure and an MHC polypeptide.The present disclosure also relates to a method for producing peptide-specific immune effector cells, comprising the steps of (a) obtaining a starting population of immune effector cells, and (b) contacting the starting population of immune effector cells with a peptide of the present disclosure, thereby producing a peptide-specific immune effector cell.The present disclosure also describes peptide-specific engineered T cells and TCRs produced by the method of the present disclosure.A further aspect relates to an in vitro isolated dendritic cell comprising the peptide, polypeptide, nucleic acid, or expression vector of the present disclosure.

[0011] Also provided is a method for predicting patient prognosis or detecting T cell response in a patient, comprising contacting a biological sample from a patient with the composition, peptide or polypeptide of the present disclosure. A further aspect relates to a peptide-specific binding molecule that binds to the peptide of the present disclosure or binds to a peptide-MHC complex. Exemplary binding molecules include antibodies, TCR mimic antibodies, scFvs, nanobodies, aptamers, and DARPINs. A related method provides a method comprising contacting a composition comprising at least one MHC polypeptide and a peptide or polypeptide of the present disclosure with a composition comprising T cells, and detecting the T cells with bound peptide and / or MHC polypeptide by detecting the detection tag.

[0012] A further aspect relates to a kit comprising the peptide, polypeptide, nucleic acid, expression vector, or composition of the present disclosure.A further method aspect relates to a method of cloning a T cell receptor (TCR), 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 immune effector cells specific for the peptide; and (d) isolating the TCR sequence from the purified immune effector cells.A further aspect relates to a method of generating cells, comprising transferring a nucleic acid or expression vector of the present disclosure into the cells.A further aspect relates to an in vitro method for generating a therapeutic T cell vaccine, comprising co-culturing T cells with a peptide of the present disclosure.

[0013] Nucleic acids of the present disclosure include those that encode the CDR regions, variable regions, engineered TCRs, polypeptides, TCR-a polypeptides, TCR-b polypeptides, peptides, polypeptides, and fusion proteins described herein. The nucleic acid may be RNA. The nucleic acid may be DNA or cDNA that encodes a peptide or polypeptide, or the complement of a peptide or polypeptide. The nucleic acid may comprise one of SEQ ID NO:1, 2, or a fragment thereof. The nucleic acid may comprise nucleotides 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 one of SEQ ID NOs:1, 2, or fragments thereof, 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. Compositions comprising the polypeptides, cells, nucleic acids, or engineered TCRs of the disclosure are also provided. A further aspect relates to a method for making engineered cells, comprising the step of transferring the nucleic acid or expression vector of the present disclosure into cells.A further aspect relates to a method for treating cancer in a subject, comprising administering to a subject in need thereof a polypeptide, a composition, a cell, a nucleic acid, or an engineered TCR.Methods also include a method for reducing tumor burden; a method for lysing cancer cells; a method for killing tumors / cancer cells; a method for increasing overall survival; a method for reducing the risk of cancer or the risk of tumor development; a method for increasing recurrence-free survival; a method for preventing cancer; and / or a method for reducing, eliminating, or reducing the spread or metastasis of cancer, comprising administering to a subject in need thereof a polypeptide, a composition, a cell, a nucleic acid, or an engineered TCR.Methods also include methods of reducing tumor burden; lysing cancer cells; killing tumors / cancer cells; increasing overall survival; preventing or reducing the risk of cancer recurrence; promoting cancer remission; increasing sensitivity to other or additional cancer treatments; reducing the risk of cancer or tumor development; increasing recurrence-free survival; preventing cancer; and / or reducing, eliminating or decreasing the spread or metastasis of cancer, comprising administering to a subject in need thereof a polypeptide, composition, cell, nucleic acid or engineered TCR.

[0014] A polypeptide or TCR-a polypeptide of the disclosure may comprise a CDR3 comprising an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:8, or 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. A polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:8. A polypeptide or TCR-b polypeptide of the disclosure may comprise 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 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 therein) sequence identity to SEQ ID NO:14. A polypeptide or TCR-b polypeptide of the disclosure may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:14. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO:8 and a TCR-b polypeptide comprising a CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0015] The polypeptide may comprise a variable region comprising CDR1, CDR2, and CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having at least 80% sequence identity to VSGLRG (SEQ ID NO:6). The variable region may comprise a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:6, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity. The variable region may comprise a CDR2 having at least 80% sequence identity to LYSAGEE (SEQ ID NO:7). The variable region may comprise a CDR2 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7, or 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 comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to, 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:4. The variable region may comprise the amino acid sequence of SEQ ID NO:4. The polypeptide may comprise a T cell receptor alpha (TCR-a) variable region. The polypeptide may comprise a TCR-a variable region and a constant region. The polypeptide may further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:5. The signal peptide may comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: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 to SEQ ID NO:5. The signal peptide may comprise the amino acid sequence of SEQ ID NO:5. The variable region may comprise CDR1, CDR2, and / or CDR3.

[0016] The variable region may comprise a CDR1 having at least 80% sequence identity to SGHDT (SEQ ID NO:12). The variable region may comprise a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:12, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:12. The variable region may comprise a CDR2 having at least 80% sequence identity to YYEEEE (SEQ ID NO:13). The variable region may comprise a CDR2 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:13, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:13. The variable region may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:12 and / or a CDR2 comprising the amino acid sequence of SEQ ID NO:13. The variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10.The variable region may comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 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 to SEQ ID NO: 10. The variable region may comprise the amino acid sequence of SEQ ID NO: 10. The polypeptide may comprise a T cell receptor beta (TCR-b) variable region. The polypeptide may comprise a TCR-b variable region and a constant region. The polypeptide may comprise, or further comprise, a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:11. The signal peptide can comprise an amino acid sequence that has 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:11, or has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:11. The signal peptide comprises the amino acid sequence of SEQ ID NO:11. The variable region can include CDR1, CDR2, and / or CDR3.

[0017] The TCR may comprise a TCR-a polypeptide comprising a variable region comprising CDR1, CDR2, and CDR3, and a TCR-b polypeptide comprising a variable region comprising CDR1, CDR2, and CDR3. The TCR-a polypeptide may comprise a CDR1 having at least 80% sequence identity to SEQ ID NO:6, and / or the TCR-b polypeptide may comprise a CDR1 having at least 80% sequence identity to SEQ ID NO:12. The TCR-a polypeptide may comprise a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:6, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:6, and / or the TCR-b polypeptide may comprise a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: It may comprise a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:12, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity. The TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:6, and the TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:12. The TCR-a polypeptide may comprise a CDR2 having at least 80% sequence identity to SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 having at least 80% sequence identity to SEQ ID NO:13.The TCR-a polypeptide may comprise a CDR2 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7, and the TCR-b polypeptide may comprise a CDR2 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:13, or comprises a CDR2 having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:13. The TCR-a polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:7, and the TCR-b polypeptide comprises a CDR2 comprising the amino acid sequence of SEQ ID NO:13. The CDR1, CDR2, and CDR3 of the TCR-a polypeptide may comprise the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide may comprise the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively. The TCR-a variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, and the TCR-b variable region comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10.The TCR-a variable region may comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:4, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:4, and the TCR-b variable region may comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:4. 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:10, or 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 to NO:10. The TCR-a polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, and the TCR-b polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:9.The TCR-a polypeptide may comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:3, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:3, and the TCR-b polypeptide may comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:3. The TCR-a and / or TCR-b polypeptides may comprise a signal peptide. The signal peptide may comprise SEQ ID NO:5 or 11. The signal peptide can comprise an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:5 or 11, 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.

[0018] The TCR may contain modifications or be chimeric. The variable region of the TCR may be fused to a TCR constant region that is different from the constant region of the cloned TCR that specifically binds the peptide of the present disclosure.

[0019] The TCR-a polypeptide and the TCR-b polypeptide may be operably linked. The term "operably linked" may refer to a covalent bond, such as a peptide bond (e.g., two elements are polypeptides and are on the same polypeptide), or a non-covalent bond, such as van der Waals forces (e.g., two polypeptides that have some degree of specific binding affinity for each other). The TCR-a polypeptide and the TCR-b polypeptide are operably linked via a peptide bond. The TCR-a polypeptide and the TCR-b polypeptide are on the same polypeptide, with the TCR-b being amino-proximal to the TCR-a. The polypeptide may be further defined as a single chain TCR. The TCR-a polypeptide and the TCR-b polypeptide may be on the same polypeptide, with the TCR-a being amino-proximal to the TCR-b. The TCR may include a linker between the TCR-a polypeptide and the TCR-b polypeptide. The linker may include glycine and serine residues. The linker may be composed exclusively of glycine and serine residues (glycine-serine linker). The linker may be a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS-SEQ ID NO:18)n, (G4S)n, and (GGGS-SEQ ID NO:19)n, 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 linkers in the polypeptides of the present disclosure.Exemplary linkers may 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), and the like. Additional linkers useful in the polypeptides and TCRs of the present disclosure are described herein. When a first region is attached to the carboxy terminus of a second region, the first region is carboxy-proximal to the second region. There may be additional intervening amino acid residues between the first and second regions. Thus, unless specifically specified as having no intervening amino acid residues, the regions need not be directly adjacent. The term "amino-proximal" is similarly defined as when a first region is attached to the amino terminus of a second region, the first region is amino-proximal to the second region. Likewise, unless otherwise specified, there may be further intervening amino acid residues between the first and second domains.

[0020] CDRs may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23 or more (or any range derivable therein) contiguous amino acid residues flanking one or both sides of a particular CDR sequence; thus, there may be one or more additional amino acids at the N-terminus or C-terminus of a particular CDR sequence, such as those shown in SEQ ID NOs: 6-8 and 12-14. Alternatively, or in combination, a CDR may also be a fragment of a CDR as described herein and 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 may be conjugated to a detection or therapeutic agent. The agent may include a fluorescent molecule, a radioactive molecule, or a toxin. The TCR or fusion protein may be conjugated to an agent described herein.

[0022] The present disclosure also provides: and / or a TCR-a polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF2024538169000010.tif4128. A nucleic acid encoding a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity to TIFF2024538169000011.tif4128 is provided. The nucleic acid may comprise a TCR-a polypeptide comprising a CDR3 comprising an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:8, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:8. 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:14, or may encode a TCR-b polypeptide comprising a CDR3 comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity. The nucleic acid may encode a TCR-a polypeptide comprising CDR1, CDR2, and CDR3, and / or a TCR-b polypeptide comprising CDR1, CDR2, and CDR3. The nucleic acid may encode a TCR-a comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:6 and / or a TCR-b comprising a CDR1 having at least 80% sequence identity to SEQ ID NO:12.The nucleic acid may comprise a TCR-a comprising a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:6, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:6. The present invention may encode a TCR-b comprising a CDR1 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:12, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity. The nucleic acid may encode a TCR-a comprising a CDR2 having at least 80% sequence identity to SEQ ID NO:7 and / or a TCR-b comprising a CDR2 having at least 80% sequence identity to SEQ ID NO:13.The nucleic acid may comprise a TCR-a comprising a CDR2 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7. The polypeptide may encode a TCR-b comprising a CDR2 having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:13, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity. The nucleic acid may encode a TCR-a variable region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4 and / or a TCR-b variable region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10.The nucleic acid may comprise a TCR-a variable region comprising an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:4, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:4. The TCR-b variable region may encode a TCR-b variable region comprising an amino acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:10, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity. The nucleic acid may 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 acid sequence of SEQ ID NO:10. The nucleic acid may comprise SEQ ID NO:1 and / or SEQ ID NO:2.The nucleic acid has 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:1, or has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:1, and / or The nucleic acid sequence may comprise a nucleic acid sequence having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:2, or at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:2.

[0023] The nucleic acid may comprise TCR-a (TRA) and TCR-b (TRB) genes. The nucleic acid may be polycistronic. The nucleic acid may also comprise an internal ribosome entry site (IRES) or a P2A linker. The nucleic acid may comprise a cDNA encoding the TCR-a and / or TCR-b genes. The nucleic acid may encode or further encode a polypeptide comprising a CD3 binding region. The CD3 binding region may comprise a CD3-specific fragment antigen binding portion (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, or 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 or consist of the amino acid sequence of SEQ ID NO:15. The peptide may be 13 amino acids in length or shorter. A peptide may have at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein), at most 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein), exactly 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein), or consist of 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein). A peptide may consist of 9 amino acids. A peptide may consist of 8 amino acids. A peptide may consist of 7 amino acids. A peptide may consist of 6 amino acids. A peptide may be defined as immunogenic. The term immunogenic may refer to the generation of an immune response, such as a protective immune response. A peptide may be modified. The modification may include conjugation to a molecule. The molecule may be an antibody, a lipid, an adjuvant, or a detection moiety (tag). The peptide may contain 100% sequence identity to the peptide of SEQ ID NO:15.The peptides of the 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 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. The peptide may have 77% sequence identity to the peptide of SEQ ID NO:15, or may have at least 77% sequence identity to the peptide of SEQ ID NO:15. The peptide may have 88% sequence identity to the peptide of SEQ ID NO:15, or may have at least 88% sequence identity to the peptide of SEQ ID NO:15. The peptides of the disclosure may have one, two, or three substitutions relative to a peptide of SEQ ID NO: 15. The peptides may have at least one, two, three, four, or five substitutions relative to a peptide of SEQ ID NO: 15, or at most one, two, three, four, or five substitutions.

[0025] The composition may be formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. The peptide may be contained in a liposome, a lipid-containing nanoparticle, or a lipid-based carrier. The composition of the present disclosure may be formulated as a vaccine. The composition may further include an adjuvant.

[0026] The dendritic cells may include mature dendritic cells. The cells may be cells having an HLA-A type. The HLA may be HLA-A, HLA-B or HLA-C. The cells may be HLA-A02 type. The cells may be HLA-A0201 type. The cells may be HLA-A01, HLA-A02, HLA-A11, HLA-A24, HLA-B07, HLA-B08, HLA-B15 or HLA-B40.

[0027] The method may further comprise isolating the expressed peptide or polypeptide. The T cells may comprise CD8+ T cells. The T cells may be 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 may contain both the TCR-a and TCR-b genes. The vector may contain a promoter that directs expression of the nucleic acid. The promoter may include a murine stem cell virus (MSCV) promoter.

[0029] The cells may comprise stem cells, progenitor cells, immune cells or natural killer (NK) cells. The cells may comprise hematopoietic stem or progenitor cells, T cells, cells differentiated from mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs). The cells may be isolated or derived from peripheral blood mononuclear cells (PBMCs). The T cells may comprise 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 may be isolated from a cancer patient. The cells may be isolated from a non-cancer patient. The cells may be isolated from a healthy patient. The cells may be frozen or unfrozen. The cells may be in cell culture. The cells may lack endogenous expression of TCR genes. The cells may further comprise a chimeric antigen receptor (CAR).

[0030] The composition may be confirmed as serum-free, mycoplasma-free, endotoxin-free, and / or sterile. The method may include, or may further include, culturing the cells in a medium, incubating the cells under conditions that allow the cells to divide, screening the cells, and / or freezing the cells.

[0031] The subject may be diagnosed with cancer, such as a cancer described herein. The cancer may include a solid tumor. The subject may have been previously treated for cancer. The subject may be determined to be resistant to a previous treatment. The method may include or further include administering an additional therapy. The cancer may be further defined as a solid tumor. The cancer may be a blood cancer, such as leukemia. The cancer may be a cancer described herein. The cancer may be stage I, stage II, stage III, or stage IV cancer. The cancer may include metastatic and / or recurrent cancer. The cancer may include a NDC80 CT antigen+ cancer. The cancer may include a cancer that expresses the peptide of SEQ ID NO:15. The subject or patient may be determined to have NDC80 CT antigen+ cancer cells or cancer (cancel) cells that are positive for the epitope of SEQ ID NO:15. The cancer may be an NDC80 CT antigen overexpressing cancer, such as overexpressed compared to a non-cancerous subject. The subject or patient may be one determined to have NDC80 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 be an experimental animal, such as a mouse, rat, rabbit, dog, cat, horse or pig. The subject may be a human subject. The subject may be a human subject, such as a human subject, having HLA-A2 and / or HLA-A * 0201 may have been determined to be positive.

[0032] The composition of the present disclosure can be formulated as a vaccine. The composition and method of the present disclosure provide prophylactic therapy to prevent cancer. The composition and method of the present disclosure provide therapeutic therapy to treat existing cancer, such as for treating patients with cancer. The composition can include or further include adjuvants. Adjuvants are known in the art and include, for example, TLR agonists and aluminum salts.

[0033] The methods of the disclosure may include, or may further include, screening the cells for one or more cellular properties, such as for TCR expression, incorporation of a nucleic acid encoding a TCR gene, or immunogenicity, such as binding of the TCR to a cancer antigen, such as the NDC80 CT antigen or a peptide of SEQ ID NO:15.

[0034] The method may include, or may further include, administering cells, including autologous cells, or a composition comprising cells. The cells may include non-autologous cells. The cells may be allogeneic or xenogeneic.

[0035] The composition may comprise the MHC polypeptides and peptides of the present disclosure, where the MHC polypeptides and / or peptides are 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 be simply detected or quantified. A simply detected response generally includes a response that simply confirms its presence, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In luminescence or fluorescence assays, the detectable response may occur directly using a luminophore or fluorophore associated with the 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 luminescence tags that generate signals include, but are not limited to, bioluminescence and chemiluminescence. Examples of suitable fluorescent tags include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th). Detection tags also include streptavidin or its binding partner, biotin.

[0036] The MHC polypeptide and peptide may be operably linked. The term "operably 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 and / or on the same polypeptide, such as a fusion protein, or the components may have some degree of binding affinity for each other, such as binding affinity caused by van der Waals forces. Thus, the MHC polypeptide and peptide may be operably linked via a peptide bond. The MHC polypeptide and peptide may be operably linked by van der Waals forces. The peptide-MHC may be operably linked to form a pMHC complex. At least two pMHC complexes may be operably linked together. Other aspects include 2, 3, 4, 5, 6, 7, 8, 9 or 10 pMHC complexes operably linked to each other, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 pMHC complexes operably linked to each other, or at most 2, 3, 4, 5, 6, 7, 8, 9 or 10 pMHC complexes operably linked to each other. At least two MHC polypeptides may be linked to one peptide. The average ratio of MHC polypeptide to peptide may be between 1:1 and 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 from about 1, 2, 3, 4, 5 or 6 to about 1, 2, 3, 4, 5 or 6 (or any range derivable therein).

[0037] The peptides can be complexed with MHC. The MHC can include HLA-A, HLA-B or HLA-C types. The 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 surfaces can include MHC polypeptides conjugated or linked to the surface. Exemplary surfaces include beads, microplates, glass slides or cell culture plates.

[0038] The method of the present disclosure may further include counting the number of T cells bound to the peptide and / or MHC. The composition comprising T cells may be isolated from a subject. The subject may be as defined herein, such as a human subject. The method may further include sorting the number of T cells bound to the peptide and / or MHC. The method of the present disclosure may also include or further include sequencing one or more TCR genes from the T cells bound to the peptide and / or MHC. The method may include or further include sequencing TCR alpha and / or beta genes from a TCR, such as a TCR that binds to a peptide of the present disclosure. The method may also include or further include grouping lymphocyte interactions by paratope hotspot (GLIPH) analysis, which 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 can further comprise culturing the cell of the present disclosure in a medium, incubating the cell under conditions that allow the cell to divide, screening the cell, and / or freezing the cell.The method can also further comprise isolating the peptide or polypeptide expressed from the cell of the present disclosure.

[0040] The disclosed method may include or further include screening the 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 may include GM-CSF. The cell characteristics may include cell surface expression of one or more of CD86, HLA, and CD14. The dendritic cells may be derived from CD34+ hematopoietic stem or progenitor cells.

[0041] The contacting step in the disclosed method may be further defined as co-culturing the starting population of immune effector cells with antigen presenting cells (APCs), where the APCs present the peptide on their surface. The APCs may be dendritic cells. The dendritic cells may be derived from peripheral blood monocytes (PBMCs). The dendritic cells may be isolated from PBMCs. The cells from which the dendritic cells or DCs are derived are isolated by leukapheresis.

[0042] A peptide-MHC (pMHC) complex of the present disclosure may be generated by contacting a peptide of the present disclosure with an MHC complex. The peptide may be expressed in a cell and bind to an endogenous MHC complex to form pMHC. A pMHC complex may be generated using peptide exchange. For example, a cleavable peptide may be designed, such as a photocleavable peptide, that binds to and stabilizes MHC. 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 disintegrates unless UV irradiation is performed in the presence of a "rescue peptide." Thus, a peptide of the present disclosure may be used as a "rescue peptide" in a peptide exchange procedure. The present disclosure also describes a pMHC complex that includes a peptide of the present disclosure. The pMHC complex may be operably linked to a solid support or may be attached to a detectable moiety, such as a fluorescent molecule, a radioisotope, or an antibody. The present disclosure also describes peptide-MHC multimeric complexes that contain at least 1, 2, 3, 4, 5 or 6 peptide-MHC molecules, or at most 1, 2, 3, 4, 5 or 6 peptide-MHC molecules, operably linked together. Linkage may be covalent, such as through a peptide bond, or non-covalent. The pMHC molecules may be bound to a biotin molecule. Such pMHC molecules may be multimerized by binding with a streptavidin molecule. The pMHC multimers may be used to detect antigen-specific T cells or TCR molecules present in a composition or tissue. The multimers may be used to detect peptides 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 slide. Cells may then be added to the slide and detection of binding between the pMHC multimers and the cells may be performed. Thus, the pMHC molecules and multimers of the present disclosure may be used to detect and diagnose cancer in a subject or to determine the immune response in an individual with cancer.

[0043] As defined in the methods described herein, obtaining may include or may further include isolating a starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs). The starting population of immune effector cells may be obtained from a subject. The method of the present disclosure may include or may further include a step of introducing a peptide or a nucleic acid encoding the peptide into dendritic cells prior to co-culture. The introduction of the peptide may be performed by transfecting or infecting dendritic cells with a nucleic acid encoding the peptide, or by incubating the peptide with dendritic cells. The peptide or the nucleic acid encoding the peptide may 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 are useful in the method of the present disclosure for transferring the nucleic acid of the present disclosure into cells. The peptide or the nucleic acid encoding the peptide may be introduced by adding the peptide or the nucleic acid encoding the peptide to dendritic cell culture medium. The immune effector cells can be co-cultured with a second population of dendritic cells transfected with a peptide or a nucleic acid encoding the peptide. 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). Clonal populations of peptide-specific immune effector cells can be generated by limiting or serial dilution followed by expansion of individual clones by rapid expansion protocols.

[0044] Purifying may further include generating a clonal population of peptide-specific immune effector cells by limiting or serial dilution of the sorted cells followed by expansion of individual clones by a rapid expansion protocol. The method of the present disclosure may include or further include cloning of T cell receptors (TCRs) from a clonal population of peptide-specific immune effector cells. The term isolation in the method of the present disclosure may be defined or further defined as cloning of T cell receptors (TCRs) from a clonal population of peptide-specific immune effector cells. Cloning of TCRs may include cloning of TCR α and β chains. TCRs may be cloned using the Rapid Amplification of 5'-cDNA Ends (RACE) method. TCR α and β chains may be cloned using the Rapid Amplification of 5'-cDNA Ends (RACE) method. The cloned TCRs may be subcloned into an expression vector. The expression vector may include a linker domain between the TCR α and TCR β sequences. The expression vector may be a retroviral or lentiviral vector. The vector may be an expression vector as described herein. The linker domain may comprise 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 TCR alpha and TCR beta sequences may be linked by an IRES sequence.

[0045] To generate engineered cells expressing the TCR α 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 +The type of T cells described herein may be T cells, CD4+ T cells, or γδ T cells. The starting population of immune effector cells may be obtained from a subject with cancer, and the host cells are allogeneic or autologous to the subject. The peptide-specific T cells may be autologous or allogeneic. A population of CD4+ or CD8+ and peptide-MHC-tetramer-positive engineered T cells may be purified from the transduced host cells. A clonal population of peptide-specific engineered T cells may be generated by limiting or serial dilution, followed by expansion of individual clones by rapid expansion protocols. In the method of the present disclosure, purifying may be defined as purifying a population of CD4+ or CD8+ 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 that is conjugated to a solid support. The solid support can include a microplate, a bead, a glass surface, a slide, or a cell culture dish. The solid support can include a nanofluidic chip. Detecting the T cell response can include or further include detecting the binding of the peptide to a T cell or TCR. Detecting the T cell response can include ELISA, ELISPOT, or tetramer assay.

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

[0048] "Treatment" or treating may refer to any treatment of a disease in a mammal, including (i) preventing the disease, i.e., preventing the onset of clinical symptoms of the disease by administration of a protective composition prior to the induction of the disease; (ii) inhibiting the disease, i.e., preventing the onset of clinical symptoms of the disease by administration of a protective composition after an inducing event but prior to the clinical appearance or reappearance of the disease; (iii) arresting the disease, i.e., preventing the onset of clinical symptoms by administration of a protective composition after their initial appearance; and / or (iv) alleviating the disease, i.e., causing regression of clinical symptoms by administration of a protective composition after their initial appearance. Treatment may exclude prevention of the disease.

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

[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 more."

[0051] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "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 can be specifically excluded from an embodiment or aspect.

[0052] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), "characterized by" (and any form of including, such as "characterized as") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0053] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this application. The phrase "consisting of" excludes any elements, steps, or components not specified. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term "comprising" can also be implemented in the context of the term "consisting of" or "consisting essentially of."

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

[0055] The use of one or more sequences or compositions can be utilized according to any of the methods described herein.Other aspects are discussed throughout this application.Any aspect or aspect discussed with respect to one aspect of this disclosure also applies to other aspects of this disclosure, and vice versa.

[0056] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the present invention may be applied to any other embodiment or aspect of the present invention.Furthermore, 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.Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, for example in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legend Descriptions.

[0057] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments and aspects of the present invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

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

[0059] [Figure 1] NDC80 expression levels in human normal tissues (GTEx database). [Diagram 2] NDC80 expression levels in human cancer tissues (TCGA database). [Diagram 3]MS Identification of NDC80 HLA-A0201 Restricted Peptides. Immunoprecipitation (IP) method was used to isolate MHC / peptides from K562-A2 cell line lysates. Peptides were eluted using acetic acid. Eluted peptides were separated by HPLC and identified by mass spectrometry (MS). One HLA-A0201 restricted peptide (GLNEEIARV - SEQ ID NO:15) from NDC80- NDC80-330 was found with high ion score (ion score = 44). [Figure 4] Generation of NDC80-330 HLA-A0201-restricted peptide (GLNEEIARV) CTL. Mature dendritic cells were pulsed with NDC80-330 (GLNEEIARV) peptide and then co-cultured with autologous PBMCs from HLA-A0201-positive healthy donors. After two rounds of stimulation, a portion of T cells was harvested from each well for tetramer detection. The tetramer+ / CD8+ population was then selected and expanded by rapid expansion protocol (REP). After 2 weeks of REP, highly pure CTL (>90% tetramer+ population) was observed. [Figure 5A] Figure 5A-J: Functional validation of NDC80-330 peptide-specific CTL cell lines. (A) T2 cells pulsed with various concentrations of NDC80-330 peptide were used as targets. The lytic ability of NDC80-330 CTL cell lines was detected by Cr51 release assay (CRA). The effector to target (E:T) ratio is 20:1. (B) Normal lung cell line HSAEC2-KT, (C-F) glioblastoma cell lines U87MG, T98G, LN-18 and M059K were used as targets to test the recognition of NDC80-330 CTL cell lines using CRA. (G-J) Pancreatic cancer cell lines PANC-1, Panc02.13, Panc03.27 and CFPAC-1 were used as targets to test the recognition of NDC80-330 CTL cell lines using CRA. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 5G] See legend to Figure 5A. [Figure 5H] See legend to Figure 5A. [Figure 5I] See legend to Figure 5A. [Figure 5J] See legend to Figure 5A. [Figure 6A] Figure 6A-D: Non-radioactive target inhibition assay to verify the recognition specificity of NDC80-330-specific CTL. 51Cr-labeled glioblastoma cell line LN-18 (A) and pancreatic cancer cell lines PANC-1, Panc03.27, and CFPAC-1 (B-D) were used as radioactive targets. T2 cells pulsed with NDC80-330 peptide without 51Cr labeling served as non-radioactive targets. T2 cells pulsed with irrelevant peptide M26 served as control non-radioactive targets. E:T is 20:1. Non-radioactive target:radioactive target is 10:1 or 20:1. Killing inhibition of non-radioactive targets against NDC80-330 CTL cell line was detected by CRA. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 7] Generation of NDC80-330-specific TCR-T. The full-length TCR was cloned into the retroviral vector pMSGV1 to generate a recombinant retrovirus. PBMCs from an HLA-A0201 healthy donor were infected with the recombinant retrovirus. After infection, the tetramer+ / CD8+ population was selected and expanded. After expansion, highly purified NDC80-330-specific TCR-T was obtained. [Figure 8A]Figure 8A-J: Functional validation of NDC80-330-specific TCR-T by killing assay. (A) T2 cells pulsed with various concentrations of NDC80-330 peptide were used as targets. The lytic ability of NDC80-330 TCR-T was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio is 20:1. (B) Immortalized normal lung cell line HSAEC2-KT, (C-F) glioblastoma cell lines U87, T98G, LN-18 and M095K, and (G-J) pancreatic cancer cell lines PANC-1, Panc02.13, Panc03.27 and CFPAC-1 were used as targets to test the recognition of NDC80-330 TCR-T using CRA. The effector-to-target (E:T) ratio is 40:1 to 1.25:1. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 8G] See legend to Figure 8A. [Figure 8H] See legend to Figure 8A. [Figure 8I] See legend to Figure 8A. [Figure 8J] See legend to Figure 8A. [Figure 9-1] Figure 9: Functional detection of NDC80-330 TCR-T by intracellular cytokine staining (ICS) assay. NDC80-330 TCR-T cell line was co-cultured with T2+M26 peptide, M2+NDC80-330 peptide, normal lung cell line HSAEC2-KT, tumor targets A375, U87MG, LN-18, T98G, M059K, PANC-1, Panc02.13, Panc03.27 and CFPAC-1 at E:T=10:1 ratio. After overnight co-culture, TCR pathway downstream activation markers CD137, CD69, IFN-γ and TNF-α were detected by ICS assay. [Figure 9-2]See description of Figure 9-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Detailed Description of the Invention The present disclosure provides a T cell receptor (TCR) that recognizes an HLA-A2 restricted epitope from the tumor antigen NDC80 CT having the amino acid sequence GLNEEIARV (SEQ ID NO:15). The present disclosure also provides a nucleotide sequence encoding the TCR, an expression vector containing the nucleotide sequence that can be used to modify cells, such as peripheral blood mononuclear cells, to generate NDC80 CT specific T cells. The present disclosure also provides the use of NDC80 CT specific T cells for adoptive immunotherapy of HLA-A2 positive cancer patients whose malignant cells express the NDC80 CT antigen.

[0061] I. Engineered T Cell Receptors The T cell receptor contains two distinct polypeptide chains, called the T cell receptor α (TCRα) and β (TCRβ) chains, linked by disulfide bonds. These α:β heterodimers are very similar in structure to the Fab fragments of immunoglobulin molecules and are responsible for antigen recognition by most T cells. A minority of T cells have a different, but structurally similar, receptor composed of a pair of distinct polypeptide chains, called γ and δ. Both types of T cell receptors differ from membrane-bound immunoglobulins that function as B cell receptors: T cell receptors have only one antigen-binding site, whereas B cell receptors have two, and T cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.

[0062] Both chains of the T cell receptor have an amino-terminal variable (V) region with homology to immunoglobulin V domains, a constant (C) region with homology to immunoglobulin C domains, and a short hinge region containing cysteine ​​residues that form interchain disulfide bonds. Each chain spans the lipid bilayer by a hydrophobic transmembrane domain and terminates in a short cytoplasmic tail.

[0063] The three-dimensional structure of the T cell receptor has been determined. This structure is certainly similar to that of an antibody Fab fragment, as inferred from previous studies of the genes encoding the T cell receptor. The chains of the T cell receptor fold almost identically to those of the Fab fragment, although the final structure appears to be a bit shorter and wider. However, there are some clear differences between the T cell receptor and the Fab fragment. The most striking difference is in the Cα domain, which folds differently from any other immunoglobulin-like domain. The half of the domain juxtaposed with the Cβ domain forms a β-sheet similar to those found in other immunoglobulin-like domains, while the other half of the domain is formed of loosely packed chains and short segments of α-helices. An intramolecular disulfide bond, which normally links two β-strands in immunoglobulin-like domains, links the β-strand to this segment of α-helix in the Cα domain.

[0064] There are also differences in the way the domains interact. The interface between the V and C domains of both T cell receptor chains is more extensive than in antibodies, which may reduce the mobility of the hinge junction between the domains. And the interaction between the Cα and Cβ domains is distinctive in that it is carbohydrate-assisted, with sugar groups from the Cα domain making several hydrogen bonds to the Cβ domain. Finally, comparison of the variable binding sites shows that the complementarity determining region (CDR) loops are aligned fairly closely with the loops of the antibody molecule, but with some displacement compared to the loops of the antibody molecule. This displacement is particularly pronounced in the Vα CDR2 loop, which is oriented at almost a right angle to the equivalent loop of the antibody V domain, as a result of a shift of the β strand from one face of the domain that anchors one end of the loop to the other. The strand displacement also causes a change in the orientation of the Vβ CDR2 loop in two of the seven Vβ domains whose structures are known. So far, the crystal structures of seven T cell receptors have been solved at this level of resolution.

[0065] Aspects of the present disclosure relate to engineered T cell receptors. The term "engineered" refers to T cell receptors with TCR variable regions grafted to TCR constant regions to create chimeric polypeptides that bind to the peptides and antigens of the present disclosure. In certain aspects, TCRs are used for cloning, enhancing expression, detection, or therapeutic control of constructs, but contain intervening sequences that are not present in endogenous TCRs, such as multiple cloning sites, linkers, hinge sequences, modified hinge sequences, modified transmembrane sequences, detection polypeptides or molecules, or therapeutic controls that may allow selection or screening of cells that contain TCRs.

[0066] In some aspects, the TCR comprises non-TCR sequences.Thus, certain aspects relate to TCRs with sequences that are not derived from TCR genes.In some aspects, the TCR is chimeric in that it comprises sequences that are normally found in TCR genes, but from at least two TCR genes that are not necessarily found together in nature.

[0067] In some aspects, the engineered TCRs of the present disclosure comprise the variables shown below: TIFF2024538169000012.tif128159TIFF2024538169000013.tif215159TIFF2024538169000014.tif171159

[0068] The following table characterizes aspects of TCR-a: TIFF2024538169000015.tif67166

[0069] The following table characterizes aspects of TCR-b: TIFF2024538169000016.tif83166

[0070] II. Proteinaceous Compositions As used herein, "protein", "peptide" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, a wild type version of a protein or polypeptide is utilized, but in many aspects of the present disclosure, a modified protein or polypeptide is utilized to generate an immune response. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is modified relative to the wild type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be modified with respect to one activity or function, but may retain wild type activity or function in other respects, such as immunogenicity.

[0071] When a protein is specifically referred to herein, it generally refers to a natural (wild type) or recombinant (modified) protein, or a protein with any signal sequence removed, if desired. A protein can be directly isolated from an organism in which it is naturally occurring, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide, but generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule.

[0072] In certain aspects, the size of the protein or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800 , 825, 850, 875, 900, 925, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acid or nucleic acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that the polypeptides may also be mutated by truncation to be shorter than their corresponding wild-type form, and may be modified by fusing or conjugating heterologous proteins or polypeptide sequences with specific functions (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).

[0073] A polypeptide, protein, or polynucleotide encoding such a polypeptide or protein of the disclosure may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more variant amino acid or nucleic acid substitutions, or may be selected from the group consisting of SEQ ID NO: NO: At least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 , 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 , 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids, or any range derivable therein, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 3, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 1 26, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 , 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids, or derivable therein The peptide or polypeptide may be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) similar, identical or homologous to any of the ranges. In certain aspects, the peptide or polypeptide is a human sequence or is based on a human sequence. In certain aspects, the peptide or polypeptide is non-naturally occurring and / or in a combination of peptides or polypeptides.

[0074] In some aspects, the protein, polypeptide or nucleic acid is selected from the group consisting of amino acids or nucleotides 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, 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).

[0075] In some aspects, the protein, polypeptide or nucleic acid is selected from the group consisting of amino acids or nucleotides 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, 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) and may include 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% relative to one of SEQ ID NOs: 1-17. (or any range derivable therein), or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) sequence identity.

[0076] In some aspects, the protein, polypeptide or nucleic acid is selected from the group consisting of SEQ ID NO: NO: 1-17, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 , 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 , 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any range derivable therein), at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 1 22, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 3, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184 , 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 2 78, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any range derivable therein), or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 10 2, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 20 6, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 31 48, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 , 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319 or 320 (or any range derivable therein) consecutive amino acids or nucleic acids.

[0077] In some aspects, a polypeptide, protein or nucleic acid has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similar to one of SEQ ID NOs: 1-17. (or any range derivable therein), at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein), or that is exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) similar, identical or homologous to a SEQ ID NO: 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, 1 73, 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, 20 4, 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, 2 98, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any range derivable therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 1 3, 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, 1 66, 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, 7, 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) contiguous amino acids.

[0078] In some aspects, any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 12 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, 5, 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, 1 46, 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, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 22 7, 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, 2 38, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 15, 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, 86 1, 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 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、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 8 68, 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, 89 9, 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), at most 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 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 6, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、 、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854、855、856、857、858、859、860、861、862、863、864、865、866、867、868、869、870、871、872、873、874、875、876、877、878、879、880、881、882、883、884、885、886、887、888、889、890、891、892、893、894、895、896、897、898、899、900、901、902、903、904、905、906、907、908、909、910、911、912、913、914、915、916、917、918、919、920、921、922、923、924、925、926、927、928、929、930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949 or 950 (or any range derivable therein), or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 11 1, 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, 1 87, 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, 21 8, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 8 58, 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, 9 In one embodiment, there is a nucleic acid molecule or polypeptide that contains 12, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949 or 950 (or any range derivable therein) consecutive amino acids or nucleotides.

[0079] Nucleotide and protein, polypeptide, and peptide sequences of various genes have been disclosed previously and can be found in recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information's Genbank and GenPept databases (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art.

[0080] It is contemplated that in the compositions of the present disclosure, there is about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition may be about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein), at least about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein) or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0081] The following is a discussion of altering the amino acid subunits of proteins to create equivalent or possibly improved second generation variant polypeptides or peptides. For example, certain amino acids can be substituted for other amino acids in a protein or polypeptide sequence with or without significant loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. Since it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and still produce a protein with similar or desirable properties. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene encoding a protein without significant loss of its biological usefulness or activity.

[0082] The term "functionally equivalent codons" is used herein to refer to codons that code for the same amino acid, such as the six different codons for arginine. "Neutral substitutions" or "neutral mutations," which refer to alterations of codons that code for biologically equivalent amino acids, are also contemplated.

[0083] The amino acid sequence variants of the disclosure can be substitution, insertion, or deletion variants. Mutations in the polypeptides of the disclosure can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more (or any range derivable therein) non-contiguous or contiguous amino acids of a protein or polypeptide compared to the wild type. Variants can include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical to any sequence provided or referenced herein, including all values ​​and ranges in between. 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.

[0084] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially identical as set forth in one of the sequences disclosed herein, so long as they meet the above criteria, including the maintenance of the biological protein activity to which expression of the protein is associated. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0085] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or several adjacent amino acids may be deleted. Stop codons may be introduced (by substitution or insertion) into the coding nucleic acid sequence to generate truncated proteins.

[0086] Insertional mutants typically involve the addition of amino acid residues at non-terminal points in the polypeptide. This can include the insertion of one or more amino acid residues. Terminal additions can also be made, and these can include fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0087] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and may be designed to modify one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, i.e., one amino acid may be replaced with an amino acid of similar chemical properties. A "conservative amino acid substitution" may involve the exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, changing 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 amino acid residues that do not occur in nature, which are usually incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted amino acid moieties.

[0088] Alternatively, the substitution may be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the replacement of one amino acid residue with a chemically different residue, such as a polar or charged amino acid replacing a non-polar or uncharged amino acid and vice versa. Non-conservative substitutions may involve the exchange of a member of one amino acid class for a member from another class.

[0089] Those skilled in the art can use well-known techniques to determine suitable variants of polypeptides as described herein. Those skilled in the art can identify suitable regions of molecules that can be changed without destroying activity by targeting regions that are not believed to be important for activity. Those skilled in the art can also identify amino acid residues and parts of molecules that are conserved between similar proteins or polypeptides. In a further aspect, regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without significantly changing biological activity or adversely affecting protein or polypeptide structure.

[0090] In making such changes, the hydropathic index of the amino acids may be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​iteratively along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids may be substituted for other amino acids having similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on hydropathic index, in certain aspects, substitutions of amino acids with hydropathic indexes within ±2 are included. In some aspects of the invention, within ±1 are included, and in other aspects of the invention, within ±0.5 are included.

[0091] It is also understood in the art that similar amino acids can be effectively substituted based on hydrophilicity.In U.S. Patent No. 4,554,101, which is incorporated herein by reference, it is stated that the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein.In certain aspects, the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigen binding, i.e., as the biological properties of the protein. These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Making changes based on similar hydrophilicity values ​​includes, in certain aspects, substitution of amino acids with hydrophilicity values ​​within ±2, in other aspects within ±1, and in still other aspects within ±0.5. In some cases, epitopes can be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted with another amino acid with a similar hydrophilicity value and still result in a biologically equivalent and immunologically equivalent protein.

[0092] Moreover, one skilled in the art can review structure-function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. In view of such comparisons, one can predict the importance of amino acid residues in the protein that correspond to amino acid residues that are important for the activity or structure of the similar protein. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0093] The skilled artisan can also analyze the three-dimensional structure and amino acid sequence in relation to its structure in a similar protein or polypeptide. In view of such information, the skilled artisan can predict the alignment of the amino acid residues of the antibody with respect to its three-dimensional structure. The skilled artisan may choose not to modify amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. In addition, the skilled artisan can generate test variants containing a single amino acid substitution at each desired amino acid residue position. These variants can then be screened using standard assays for binding and / or activity, thus obtaining information gathered from such routine experiments, which allows the skilled artisan to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0094] In some aspects of the present invention, amino acid substitutions are made that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) modify binding affinity to form protein complexes, (4) modify ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in that portion of an antibody that is outside the domain that forms intermolecular contacts. In such aspects, conservative amino acid substitutions that do not substantially change the structural features of a protein or polypeptide (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes a natural antibody) can be used.

[0095] III. Nucleic acids In certain aspects, the nucleic acid sequence can be present in various instances, such as: an isolated segment of an integrated sequence or recombinant polynucleotide and a recombinant vector encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; a polynucleotide sufficient for use as a hybridization probe, PCR primer, or sequencing primer for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; an antisense nucleic acid for inhibiting expression of a polynucleotide; and a complementary sequence of the foregoing described herein. Nucleic acids encoding epitopes for certain antibodies provided herein are also provided. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0096] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from total genomic nucleic acid. Included within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in some aspects, includes a regulatory sequence that is substantially isolated from a naturally occurring gene or protein coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present in a polynucleotide.

[0097] In this regard, the terms "gene," "polynucleotide," or "nucleic acid" are used to refer to a nucleic acid (including any sequences required for proper transcription, post-translational modification, or localization) that encodes a protein, polypeptide, or peptide. As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide can include a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide can be encoded by a nucleic acid that includes variants that have slightly different nucleic acid sequences but still encode the same or substantially similar proteins.

[0098] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; the variants include at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values ​​and ranges between, compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis with standard parameters). In some aspects, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and more identity to the amino acid sequences described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0099] Regardless of the length of the coding sequence itself, the nucleic acid segment may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and therefore the total length may vary considerably. The 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 include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. It is therefore contemplated that nucleic acid fragments of almost any length can be used, with the total length being preferably limited by the ease of purification and by the intended use in the recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or to allow therapeutic utility such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0100] A. Hybridization A nucleic acid that hybridizes to another nucleic acid under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. For example, see Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those that use a hybridization temperature of 42°C and contain about 50% formamide), and washing conditions of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions involve hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash 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 will typically remain hybridized to each other.

[0101] Parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are described, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated by reference in their entireties for all purposes), and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0102] B. Mutation Changes can be introduced into nucleic acid by mutation, thereby resulting in changes in the amino acid sequence of the polypeptide (e.g., antibody or antibody derivative) that the nucleic acid encodes. Any technique known in the art can be used to introduce mutations. In one aspect, one or more specific amino acid residues are changed, for example, using a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. However created, mutant polypeptides can be expressed and screened for desired properties.

[0103] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively alters the biological activity of the polypeptide it encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, reducing, or eliminating activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0104] C. Probe In another aspect, the nucleic acid molecule is suitable for use as a primer or hybridization probe for the detection of a nucleic acid sequence. The nucleic acid molecule can comprise a portion of a nucleic acid sequence encoding a full-length polypeptide, e.g., a fragment that can be used as a probe or primer, or only a fragment that encodes an active portion of a given polypeptide.

[0105] 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 the nucleic acid molecule PCR primer can be used to amplify a region of DNA that can be used to isolate a nucleic acid sequence for use in the production of a variable domain of an antibody, among others. See, for example, Gaily Kivi et al., BMC Biotechnol. 16:2 (2016). In a preferred aspect, the nucleic acid molecule is an oligonucleotide. In a more preferred aspect, the oligonucleotide is derived from the highly variable regions of the heavy and light chains or alpha and beta chains of the antibody or TCR of interest. In an even more preferred aspect, the oligonucleotide encodes all or part of one or more CDRs or TCRs.

[0106] A probe based on the desired sequence of the nucleic acid can be used to detect the nucleic acid or a similar nucleic acid, for example, a transcript that encodes a polypeptide of interest. The probe can contain a label group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such a probe can be used to identify cells that express the polypeptide.

[0107] IV. Polypeptide Expression In some aspects, there are nucleic acid molecules (e.g., TCR genes) that encode the polypeptides or peptides of the present disclosure. These can be made by methods known in the art, such as by isolating from B cells of immunized and isolated mice, expressing them as phage displays in any suitable recombinant expression system, and allowing them to assemble to form antibody molecules, or by recombinant methods.

[0108] A. Expression The nucleic acid molecule may be used to express large amounts of the polypeptide. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule may be used for humanization of the TCR gene.

[0109] B. Vector In some aspects, expression vectors containing nucleic acid molecules encoding a polypeptide of 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 may encode heavy chains, light chains, alpha chains, beta chains, or antigen-binding portions thereof. In some aspects, expression vectors containing nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0110] To express the polypeptide or peptide of the present disclosure, the 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, the vector encodes a functionally complete human CH or CL immunoglobulin or TCR sequence with suitable restriction sites engineered so that any variable region sequence can be easily inserted and expressed. In some aspects, the vector encodes a functionally complete human TCR alpha or TCR beta sequence with suitable restriction sites engineered so that any variable sequence or CDR1, CDR2 and / or CDR3 can be easily inserted and expressed. Typically, the expression vector used in any of the host cells contains sequences for plasmid or virus maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences," typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding an expressed polypeptide, and a selectable marker element. Such sequences and methods for their use are well known in the art.

[0111] C. Expression Systems There are many expression systems that contain at least some or all of the above expression vectors. Prokaryotic and / or eukaryotic based systems can be used in one aspect to produce the nucleic acid sequence, or its cognate polypeptide, protein and peptide. Commercially and widely available systems include, but are not limited to, bacterial, mammalian, yeast and insect cell systems. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of expressed foreign proteins. Those skilled in the art can express the vector using a suitable expression system to produce the nucleic acid sequence, or its cognate polypeptide, protein or peptide.

[0112] V. Methods of Gene Transfer Suitable methods for nucleic acid delivery to result in expression of the composition are expected to include virtually any method by which 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 known to those of skill in the art. Such methods include microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference), by injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by the use of DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al., each of which is incorporated herein by reference);, 1990; U.S. Patent Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); direct delivery of DNA, such as by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0113] A. host cell In another aspect, the use of a host cell into which a recombinant expression vector has been introduced is contemplated. Antibodies can be expressed in a variety of cell types. An expression construct encoding an antibody can be transfected into a cell 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 may utilize control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain aspects, an antibody expression construct can be placed under the control of a promoter associated with T cell activation, such as one controlled by NFAT-1 or NF-κB, both transcription factors that can be activated upon T cell activation. Control of antibody expression allows T cells, such as tumor-targeting T cells, to sense their surroundings and perform real-time regulation of cytokine signaling in both the T cell itself and surrounding endogenous immune cells. Those skilled in the art will understand the conditions under which host cells are incubated to maintain them and allow replication of the vector. Techniques and conditions that allow large-scale production of vectors, as well as production of the nucleic acid encoded by the vector and its cognate polypeptide, protein, or peptide, are also understood and known.

[0114] It is known that for stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of cells can integrate foreign DNA into their genome.To identify and select these integrants, a selectable marker (e.g., for antibiotic resistance) is generally introduced into host cells together with the gene of interest.The cells that have stably transfected the introduced nucleic acid can be identified by drug selection (e.g., the cells that have integrated the selectable marker gene survive, while other cells die), among other methods known in the art.

[0115] B. Isolation The nucleic acid molecules encoding the heavy, light, alpha, and beta chains of an antibody or TCR or either or both of their variable regions can be obtained from any source that produces antibodies. Methods for isolating mRNA encoding an antibody are well known in the art. See, for example, Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, for example, Kabat et al., 1991, supra. The nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed in cells into which they have been introduced, and the antibody can be isolated.

[0116] VI. Further Therapies A. Immunotherapy In some aspects, the method includes administering an additional therapy. In some aspects, the additional therapy includes cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be classified as active, passive, or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAA); they are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapies are known in the art, and some are described below.

[0117] 1. Checkpoint Inhibitors and Combination Treatments Aspects of the disclosure can include administration of immune checkpoint inhibitors, which are further described below.

[0118] a. PD-1, PDL1, and PDL2 inhibitors PD-1 can act in the tumor microenvironment where T cells encounter infection or tumor. Activated T cells upregulate PD-1 and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 in epithelial and tumor cells. PDL2 is expressed in 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 immune responses. The inhibitors of the present disclosure can block one or more functions of PD-1 and / or PDL1 activity.

[0119] 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.

[0120] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another aspect, the PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its ligand binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its ligand binding partner. In certain aspects, the PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, as described in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0121] 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 or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some aspects, the PDL1 inhibitor comprises AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0122] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor, such as rHIgM12B7.

[0123] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Thus, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding to the same epitope on PD-1, PDL1, or PDL2 as the aforementioned antibody, and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the aforementioned antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) of variable region amino acid sequence identity to the aforementioned antibody.

[0124] b. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. Activation of T cells through T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitor 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 CTLA-4 and B7-1 interaction. In some aspects, the inhibitor blocks CTLA-4 and B7-2 interaction.

[0125] 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.

[0126] Anti-human-CTLA-4 antibodies (or VH domains and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be made using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; also known formerly as ticilimumab), U.S. Pat. No. 6,207,156; Hurwitz et al., 1998 can be used in the methods disclosed herein. The disclosure of each of the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0127] An additional anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0128] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies, and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) of variable region amino acid sequence identity to the aforementioned antibodies.

[0129] 2. Inhibition of costimulatory molecules In some aspects, the immunotherapy includes inhibitors of costimulatory molecules. In some aspects, the inhibitors include inhibitors of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0130] 3. Dendritic cell therapy Dendritic cell therapy induces anti-tumor responses by having dendritic cells present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating them to kill other cells presenting the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help target cancer antigens. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0131] One way to induce dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small portions of proteins that correspond to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to boost immune and antitumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0132] Dendritic cells can also be activated in vivo by expressing GM-CSF in tumor cells, which can be accomplished by genetically engineering the tumor cells to produce GM-CSF or by infecting the tumor cells with an oncolytic virus that expresses GM-CSF.

[0133] Another strategy is to remove dendritic cells from the patient's blood and activate them ex vivo. The dendritic cells are activated in the presence of tumor antigens, which can be single tumor-specific peptides / proteins or tumor cell lysates (a solution of destroyed tumor cells). These cells (with optional adjuvants) are injected to elicit an immune response.

[0134] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, inducing dendritic cells to mature and provide immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

[0135] 4. CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine immune cells with new specificities to target cancer cells. Usually, these receptors transfer the specificity of a monoclonal antibody to a T cell. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to the treatment using such transformed cells for cancer treatment.

[0136] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to 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. Once a T cell is engineered to become a CAR-T cell, it acts as a "living drug". CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). A key aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of the molecule that is targeted.

[0137] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some aspects, the CAR-T therapy targets CD19.

[0138] 5. Cytokine therapy Cytokines are proteins produced by many types of cells present in tumors. They can modulate the immune response. Tumors often use cytokines to promote tumor growth and reduce immune responses. These immunomodulatory effects allow them to be used as drugs to elicit an immune response. Two commonly used cytokines are interferons and interleukins.

[0139] Interferons are produced by the immune system. They are usually involved in antiviral responses, but are also used in cancer. They are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ).

[0140] Interleukins have a number of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0141] 6. Adoptive T cell therapy Adoptive T cell therapy is a form of passive immunization by transfusion of T cells (adoptive cell transfer). T cells are found in blood and tissues and are usually activated when they find a foreign pathogen. Specifically, T cells become activated when their surface receptors encounter cells that present a portion of a foreign protein on their surface antigen. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal tissues and in 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, preventing immune-mediated tumor death.

[0142] Several methods have been developed to produce and obtain tumor-targeted T cells. T cells specific for tumor antigens can be removed from tumor samples (TIL) or filtered from the blood. Subsequent activation and culture is performed ex vivo, followed by reinfusion. Activation can be achieved through gene therapy or by exposing the T cells to tumor antigens.

[0143] B. Chemotherapy In some aspects, the additional therapy comprises chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) Natural products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), and (d) various agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adrenal cortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0144] Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain aspects, effective doses used in clinical applications include about 15 mg / m2 to about 20 mg / m2 for 5 days every 3 weeks for a total of 3 courses are contemplated. In some aspects, the amount of cisplatin delivered to cells and / or subjects in conjunction with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount that would be delivered if cisplatin was used alone.

[0145] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector and doxorubicin has been found to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combined treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0146] Doxorubicin is poorly absorbed and is preferably administered intravenously.In certain aspects, suitable intravenous doses for adults include about 60 mg / m2 to about 75 mg / m2 at intervals of about 21 days, or about 25 mg / m2 to about 30 mg / m2 on each of two or three consecutive days repeated at intervals of about 3 weeks to about 4 weeks, or about 20 mg / m2 once a week.The lowest dose should be used in elderly patients when there is previous bone marrow suppression or neoplastic bone marrow infiltration caused by previous chemotherapy, or when the drug is combined with other myelopoiesis suppressing drugs.

[0147] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards may include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN® available from Mead Johnson, NEOSTAR® available from Adria) is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, about 40 mg / kg to about 50 mg / kg in divided doses for about 2 to about 5 days initially, or about 10 mg / kg to about 15 mg / kg every about 7 to about 10 days, or about 3 mg / kg to about 5 mg / kg twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal effects, the intravenous route is preferred. Drugs may also be administered intramuscularly, by infiltration, or into body cavities.

[0148] Further suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU can be administered to a subject at a dosage anywhere from about 7.5 to about 1000 mg / m2. Furthermore, 5-FU dosing schedules can be for various periods, for example, up to 6 weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0149] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and therefore would be useful in the present disclosure for these cancers as well.

[0150] The amount of chemotherapeutic agent delivered to the patient can be variable. In one suitable aspect, the chemotherapeutic agent can be administered in an amount effective to cause the arrest or regression of cancer in the host when the chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent can be administered anywhere from 2 to 10,000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. For example, the chemotherapeutic agent can be administered in an amount about 20 times less, about 500 times less, or even about 5000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct and for determining effective dosages. 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.

[0151] C. Radiation Therapy In some aspects, the additional or previous therapy comprises radiation, such as ionizing radiation.As used herein, "ionizing radiation" refers to radiation that comprises particles or photons that have sufficient energy or can produce sufficient energy through nuclear interaction to produce ionization (gain or loss of electrons).An exemplary and preferred ionizing radiation is x-rays.Means for delivering x-rays to target tissue or cells are well known in the art.

[0152] D. Surgery In some aspects, the additional therapy includes surgery. Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used with other therapies, such as treatments of aspects of the invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0153] Removal of part or all of the cancer cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be performed by perfusion, direct injection, or local application of additional anti-cancer therapy to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or any range derivable therein). These treatments may also be at various dosages.

[0154] VII. Detection and Therapeutic Agents In some aspects of the present disclosure, it will be useful to detectably or therapeutically label the TCR or fusion protein of the present disclosure.Methods for conjugating polypeptides to these agents are known in the art.For illustrative purposes only, polypeptides can be labeled with detectable moieties such as radioactive atoms, chromophores, fluorophores, etc.Such labeled polypeptides can be used in diagnostic techniques in vivo or in isolated test samples or in the methods described herein.

[0155] As used herein, the term "label" contemplates a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to a composition to be detected, e.g., a protein such as a polynucleotide or an antibody, to produce a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP). The label may be detectable itself (e.g., radioisotope label or fluorescent label) or, in the case of an enzyme label, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A simply detected response generally includes a response that simply confirms its presence, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In luminescent or fluorescent assays, the detectable response can occur directly, by means of a luminophore or fluorophore associated with the assay component actually responsible for binding, or indirectly, by means of a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0156] Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally involves a change in or the occurrence of a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th(ed.) Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0157] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th (ed.).

[0158] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of a cell or tissue, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group of the fluorescent label will depend on the site of attachment, either to the linker, agent, marker, or second labeling agent.

[0159] The attachment of the fluorescent label may be direct to the cellular component or compound, or alternatively via a linker. Suitable binding pairs for indirectly linking the fluorescent label to an intermediate include, but are not limited to, antigen / polypeptide, such as rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0160] Coupling of the polypeptide to a low molecular weight hapten can increase the sensitivity of the antibody in an assay. The hapten can then be specifically detected by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten polypeptides. See Harlow and Lane (1988), supra.

[0161] The conjugated agent can be linked to the polypeptide directly or indirectly using any of a number of available methods. For example, the agent can be attached to the hinge region of a reduced antibody component via disulfide bond formation using a cross-linking agent such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety in the Fc region of the antibody (Yu et al., 1994; Upeslacis et al., 1995; Price, 1995).

[0162] 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).

[0163] The polypeptides of the present disclosure or antigen-binding regions thereof can be linked to another functional molecule, such as a ligand, a cytotoxic molecule, a chemotherapeutic agent, or other agent described as an additional therapeutic substance.

[0164] VIII. Cell Formulation and Culture In certain aspects, the cells of the present disclosure may be specially formulated and / or they may be cultured in a specific medium. The cells may be formulated in such a way that they are suitable for delivery to a recipient without adverse effects.

[0165] In certain aspects, the medium may be prepared using a medium used to culture animal cells as the basal medium, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, 199 Medium, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fisher's medium, and any combination thereof, but the medium may not be particularly limited thereto, as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0166] The medium can be serum-containing or serum-free, or xeno-free. From the aspect of preventing contamination by heterologous animal-derived components, serum can be derived from the same animal as that of stem cells. Serum-free medium refers to a medium that does not have raw or unpurified serum, and therefore can include medium that has 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. Serum substitutes may include materials that suitably contain albumin (such as albumin substitutes such as lipid-rich albumin, bovine albumin, recombinant albumin or humanized albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereof. Serum substitutes may be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679, which is incorporated herein in its entirety. Alternatively, for more convenience, any commercially available material may 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 contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of the following: biotin; DL-alpha tocopherol acetate; DL-alpha-tocopherol; vitamins such as vitamin A (acetate); BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; proteins such as superoxide dismutase; corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or other components such as T3 (triiodo-I-thyronine). In certain aspects, one or more of these may be explicitly excluded.

[0169] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, vitamin B12 (and any range derivable therein), or the medium comprises a combination thereof or a salt thereof. In some aspects, the medium comprises or essentially consists of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamin comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises a protein. In some aspects, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, simple sugars, inorganic ions.In some aspects, the amino acid comprises arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof.In some aspects, the inorganic ion comprises sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof.In some aspects, the medium further comprises one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein, and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain aspects, one or more of these may be explicitly excluded.

[0170] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, of which in certain aspects one or more may be explicitly excluded.

[0171] One or more of the medium components has a concentration of at least 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein, and at most 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 It may be added at a concentration of 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 specially formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, and certain formulations include 2.5% human albumin. The cells may be specially formulated for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In certain aspects, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from the time of thawing.

[0173] In certain aspects, the cells of the present disclosure include an exogenous TCR, which may be of defined antigen specificity. In some aspects, the TCR can be selected based on lack or reduced alloreactivity to the intended recipient (examples include certain virus-specific TCRs, xenospecific TCRs, or cancer testis antigen-specific TCRs). In examples where the exogenous TCR is non-allo-reactive, during T cell differentiation, the exogenous TCR suppresses rearrangement and / or expression of the endogenous TCR locus through a developmental process called allelic exclusion, resulting in T cells that only express non-allo-reactive exogenous TCRs and thus become non-allo-reactive. In some aspects, the selection of the exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some aspects, the endogenous TCR gene is modified by genome editing so as not to express the protein. Methods of gene editing, such as those 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 the 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), 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, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEM, Carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, Laminin receptor, HPV Examples of antigens that may be used include E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). CARs may be first, second, third, or higher generation CARs. CARs may be bispecific for any two non-identical antigens, or specific for three or more non-identical antigens.

[0175] IX. Administration of Therapeutic Compositions The therapy provided herein may include the administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapy may be administered in any suitable manner 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] Aspects of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapies can be administered in one composition, or in two or more compositions, such as two, three or four compositions. Various combinations of agents can be utilized.

[0177] The therapeutic compositions of the present disclosure may be administered by the same or different routes of administration. In some aspects, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0178] Treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount administered, as well as the specific route and formulation, is within the skill of those in the clinical arts to determine. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some aspects, a unit dose includes a single administrable dose.

[0179] Precise amounts of a therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dosage include the physical and clinical condition of the patient, the route of administration, the intended treatment goal (alleviation of symptoms vs. cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy the subject may be receiving.

[0180] Cancers suitable for treatment include, but are not limited to, tumors of all types, locations, sizes and characteristics. In some aspects, the cancer comprises a solid tumor. In some aspects, the method relates to reducing tumor volume or treating 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 cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, pediatric cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumor brain tumor, visual pathway and hypothalamic glioma, breast cancer, lymphatic system tumor, bronchial adenoma / carcinoid, tracheal cancer, lung cancer, Burkitt's lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal cancer of unknown primary, lymphoma of the central nervous system, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's, childhood extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanomaEye cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors: extracranial, extragonadal or ovarian, gestational trophoblastic tumors, brain stem gliomas, pediatric cerebral astrocytoma, pediatric visual pathway and hypothalamic gliomas, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, pediatric intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphoblastic (also called acute lymphocytic) leukemia, acute myeloid (acute myelogenous) leukemia leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), chronic myelogenous (also called chronic myeloid leukemia), hairy cell cancer 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 of all lymphomas other than Hodgkin) lymphoma, primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma of childhood, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma of adults, mesothelioma of children, metastatic cervical squamousNeck cancer, oral cavity (mouth) cancer, multiple endocrine neoplasia, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia of adults, acute myeloid leukemia of children, multiple myeloma, chronic myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (superficial epithelial and stromal tumors), ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, islet cell sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pediatric pituitary adenoma, plasma cell neoplasms / multiple myeloma, pleuropulmonary It is suitable for treating blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, pediatric salivary gland carcinoma sarcoma, Ewing's sarcoma family of tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine Sezary syndrome sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, Merkel cell small cell lung cancer, cancer of the small intestine, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma of unknown primary, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, pediatric T-cell lymphoma, testicular cancer, throat cancer, thymoma, pediatric thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma of the endometrium, vaginal cancer, glioma of the visual pathway and hypothalamus, pediatric vulvar cancer, and Wilms' tumor (kidney cancer).

[0181] X. Kit Certain aspects of the present invention also relate to kits comprising the disclosed compositions or compositions for carrying out the methods of the present invention. In some aspects, the kits can be used to assess one or more biomarkers or HLA types. In certain aspects, the kits include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more profiles. 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, 120, 121, 122, 133, 144, 150 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any values ​​or ranges and combinations derivable therein.

[0182] The kit may include components that may be individually packaged or disposed in containers, such as tubes, bottles, vials, syringes, or other suitable container means.

[0183] Individual components may be provided in the kit in concentrated amounts; in some aspects, components are provided individually at the same concentration as they are in solution with the other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.

[0184] In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. Additionally, the kits can include samples that are negative or positive controls for methylation of one or more biomarkers.

[0185] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different aspects may be combined. It is contemplated that the claims as originally filed will cover any claim that is multiple dependent on any of the claims or combinations of the claims as filed. EXAMPLES

[0186] XI. Working Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will recognize that the procedures disclosed in the following examples are procedures that the inventors have found to work well in the practice of the present disclosure and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art will recognize in light of the present disclosure that many changes may be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0187] Example 1: Generation of NDC80-330 peptide (GLNEEIARV) antigen-specific CTL and development of TCR-T To date, we have established a successfully designed system that can enrich, isolate, and expand autologous antigen-specific T cells from peripheral blood using tetramer-induced sorting and the rapid expansion protocol (REP). If defined T cell epitopes have been identified, we can generate the patient's own antigen-specific T cells ex vivo and use this system to reveal the prerequisites for effective immune-based therapy within 6-8 weeks. Furthermore, by manipulating immune-regulatory parameters, we can enhance the effector function and long-term persistence of these expanded antigen-specific T cells in vivo. Based on the identified HLA-restricted NDC80 peptides and corresponding functional CTLs, as well as TCR-engineered T cells (TCR-T), we plan to evaluate the safety and efficacy of adoptive T cell therapy targeting the NDC80 antigen for patients with advanced or recurrent cancer in a nonrandomized dose-finding phase I / II study. Based on the results obtained so far, antigen-specific CTL or TCR-T against NDC80 peptide will be generated and expanded using the patient's own peripheral blood mononuclear cells (PBMC). After functional detection (phenotype, killing capacity, etc.), T cells will be infused into the patient in a dose-escalating strategy to evaluate safety. If no severe dose-limiting toxicity (DTL) occurs, antitumor efficacy will be further determined by staging evaluation and recorded as complete response (CR), partial response (PR) or progressive disease (PD) (RECIST criteria). During this period, the duration of the transferred CTL will be determined. Progression-free survival will also be determined, although it is not a primary endpoint in phase I trials. If no severe side effects occur and major clinical responses are observed, we plan to apply phase II trials to further evaluate the efficacy of adoptive T cell therapy using a larger cohort of patients with advanced cancer.

[0188] All of the methods disclosed and claimed herein can be made and carried out without undue experimentation in light of the present invention. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. An engineered T cell receptor (TCR) comprising a TCR-α polypeptide and a TCR-β polypeptide, wherein the TCR-α polypeptide comprises CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 6, 7, and 8, respectively, and the TCR-β polypeptide comprises CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 12, 13, and 14, respectively.

2. (a) The TCR-a polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:4, and the TCR-b polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:10, or (b) The manipulated TCR according to claim 1, wherein the TCR-a polypeptide comprises the amino acid sequence of SEQ ID NO:4 and the TCR-b polypeptide comprises the amino acid sequence of SEQ ID NO:

10.

3. The manipulated TCR according to claim 1, which is a single-stranded TCR.

4. A fusion protein comprising an manipulated TCR according to claim 1 and a CD3-binding region, wherein the CD3-binding region comprises a CD3-specific fragment antigen-binding site (Fab), a single-strand variable fragment (scFv), a single-domain antibody, or a single-strand antibody.

5. One or more polypeptides comprising the manipulated TCR according to Claim 1.

6. The polypeptide according to claim 5, further comprising a signal peptide, wherein the signal peptide comprises an amino acid sequence of SEQ ID NO:11, or an amino acid sequence having at least 80% identity with SEQ ID NO:

11.

7. One or more nucleic acids encoding the polypeptide described in claim 5.

8. (a) is RNA, or (b) DNA or cDNA encoding the polypeptide or a complement of the polypeptide, or (c) SEQ ID NO: Having at least 70% sequence identity with respect to one of the following: The nucleic acid according to claim 7.

9. A cell comprising the nucleic acid described in Claim 7.

10. The cells according to claim 9, comprising stem cells, progenitor cells, immune cells, natural killer (NK) cells, hematopoietic stem cells or hematopoietic progenitor cells, T cells, cells differentiated from mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs).

11. The cell is a T cell, and the T cell is a cytotoxic T lymphocyte (CTL), CD8 + T cells, CD4 + The cell according to claim 10, which is a T cell, an immutable NK T (iNKT) cell, a gamma-delta T cell, an NKT cell, or a regulatory T cell.

12. A composition comprising (i) an engineered TCR according to any one of claims 1 to 3, (ii) a fusion protein according to claim 4, (iii) a polypeptide according to claim 5 or 6, or (iv) a nucleic acid according to claim 7 or 8.

13. The composition according to claim 12, for use in a method for treating or preventing cancer in a subject, or for use in a method for stimulating an immune response in a subject.

14. (a) The subject is a human subject, and / or (b) The cells are autologous, and / or (c) The cells are of the same species, and / or (d) The cancer is NDC80 CT antigen positive, and / or (e) The subject is, （i） (A) Determined to have NDC80 CT antigen-positive cancer cells, and / or (B) HLA-A2 positive, and / or determined to be HLA-A2 positive, (ii) (A) Cancer cells that have been determined to express the peptide of SEQ ID NO:15, and / or (B) HLA-A2 positive, and / or determined to be HLA-A2 positive, (iii) being HLA-A0201 positive and / or having been determined to be HLA-A0201 positive, The composition for use according to claim 13.

15. A method for producing engineered cells, comprising the step of transferring the nucleic acid according to claim 7 or 8 into cells in vitro or ex vivo.

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