Methods and compositions for treating cancer

Abstract

A nucleic acid encoding: (i) a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR); and (ii) a TNFc polypeptide or functional fragment thereof is disclosed. Also described are nucleic acids encoding the receptor and polypeptide, and cells or populations of cells comprising a nucleic acid or polypeptide. Methods disclosed include methods of making a cell comprising introducing into a cell a nucleic acid, and a method of treating a patient with cancer comprising administering to the patient an effective amount of a composition comprising the nucleic acid, or the cells.

Description

METHODS AND COMPOSITIONS FOR TREATING CANCER BACKGROUND

[0001] This application claims priority of U.S. Provisional Patent Application No.63 / 769,458, filed March 10, 2025, and U.S. Provisional Patent Application No. 63 / 897,000, filed October 10, 2025, which are hereby incorporated by reference in their entirety.

[0002] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on March 9, 2026, is named UCLAP0199WO, and is 117,799 bytes in size.I. Field of the Invention

[0003] This invention relates to the field of immunology and cancer therapeutics.II. Background

[0004] (TCR) is revolutionizing cancer treatment, with dramatic clinical responses seen in a variety of solid tumors. However, while these treatments lead to objective clinical responses in many patients, a significant number of patients remain who do not respond to therapy. The identification of biological factors which contribute to the functional capacity in cellular therapies, and how these factors differ between responders and non-responders, represents an unmet need in cancer immunotherapy.SUMMARY

[0005] Described herein is a nucleic acid encoding: (i) a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR); and (ii) a TNFa polypeptide or functional fragment thereof. Also described are polypeptides expressed from the nucleic acids of the disclosure, and cells or populations of cells comprising a nucleic acid or polypeptide of the disclosure. Cell(s) also include cell(s) that comprise a heterologous nucleic acid encoding a TNFa polypeptide. Further described is a composition comprising cells of the disclosure. The composition may be in a pharmaceutically acceptable formulation. Methods include methods of making a cell comprising introducing into a cell a nucleic acid of the disclosure. Also described is a method of treating a patient with cancer comprising administering to the patient an effective amount of a composition of the disclosure. Further methods provide for a method302139246.1 - 1 -for increasing anti-tumor immunity and / or tumor cell cytotoxicity in a patient having cancer, the method comprising administering to the patient an effective amount of a composition of the disclosure.

[0006] The nucleic acid may be DNA. The nucleic acid may be RNA. The nucleic acid may be an expression construct. The expression construct may include or exclude a plasmid or viral vector. The viral vector may include or exclude a vector derived from a retrovirus or a vector derived from a lentivirus. Cells of the disclosure include or exclude those in which the viral vector has integrated into the cell’s genome.

[0007] The TNFa polypeptide may comprise the amino acid sequence of SEQ ID NO:1. The TNFa polypeptide may comprise an amino acid sequence that is a fragment of SEQ ID NO:1. The TNFa polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or to a fragment of SEQ ID NO: 1. The TNFa polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 1. The TNFa polypeptide may comprise the amino acid sequence of SEQ ID NO: 73. The TNFa polypeptide may comprise an amino acid sequence that is a fragment of SEQ ID NO:73. The TNFa polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:73 or to a fragment of SEQ ID NO:73. The TNFa polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:73.

[0008] The nucleic acid may further encode for a suicide gene product or exclude a suicide gene product. The cell may further comprise a nucleic acid encoding for a suicide gene product or the cell may exclude a nucleic acid encoding for a suicide gene. The suicide gene product may include or exclude herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypetidase G2, cytochrome P450, linamarase, P-lactamase, nitroreductase (NTR), carboxypeptidase A, split Cas9, truncated EGFR, or inducible caspase 9. The nucleic acid may further encode for a suicide gene product. Suicide gene products provide for a safety switch that can be activated to eliminate the cell from the body in case of adverse reactions. A suicide gene may be enzyme-based, meaning the gene product of the suicide gene is an enzyme and the suicide function depends on enzymatic activity. One or more suicide genes may be utilized in a single cell or clonal population. The302139246.1 - 2 -suicide gene may encode or may exclude herpes simplex virus thymidine kinase (HSV-TK), truncated EGFR, purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypetidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Methods in the art for suicide gene usage may be employed, such as in U.S. Patent No. 8628767, U.S. Patent Application Publication 20140369979, U.S. 20140242033, and U.S. 20040014191, all of which are incorporated by reference in their entirety. Caspase suicide genes are described in Di Stasi A, et al., N Engl J Med. 2011 Nov 3 ;365(18): 1673-83, which is incorporated by reference. Using truncated EGFR as a safety switch is further described in Kao RL, et al., Hum Gene Ther. 2019 Apr;30(4):413-428, which is incorporated by reference. The TK gene may be a viral TK gene, .i.e., a TK gene from a virus. The TK gene may be a herpes simplex virus TK gene. The suicide gene product may be activated by a substrate. Thymidine kinase is a suicide gene product that is activated by ganciclovir, penciclovir, or a derivative thereof. The suicide gene product may be encoded by the same or a different nucleic acid molecule encoding for the CAR and / or other components listed throughout. In alternative embodiments, the cell does not express an exogenous suicide gene.

[0009] The nucleic acid comprises one or more promoter(s) that direct the expression of the suicide gene product, CAR or TCR, and / or TNFa polypeptide. The promoter may be constitutive. The promoter may be conditional. The TNFa polypeptide may be a human TNFa polypeptide and / or derived from a human TNFa polypeptide. The TNFa may be genomically identical to the endogenous gene. The nucleic acid sequence of the TNFa may include the wildtype DNA sequence of the exons of the human TNFa DNA sequence. The DNA sequence of the TNFa may include the wild-type DNA sequence of the exons of the mouse TNFa DNA sequence. The nucleic acid may include or exclude a signal sequence. The nucleic acid may encode for a signal sequence that is at the amino-terminus of the TNFa polypeptide, CAR, and / or TCR of the polypeptide expressed from the nucleic acid. The TNFa polypeptide, CAR, and / or TCR may be expressed from one nucleic acid. The nucleic acid may express a polypeptide comprising the TNFa polypeptide, CAR, and / or TCR. The nucleic acid may include or exclude DNA. The nucleic acid may include or exclude RNA.

[0010] The nucleic acid that encodes for the TNFa polypeptide may comprise SEQ ID NO: 103 or a nucleic acid sequence that has at least 70% sequence identity to SEQ ID NO: 103. The nucleic acid encoding for the TNFa polypeptide may comprise about, or may comprise at least, or may comprise exactly, or may comprise 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, 94,302139246.1 - 3 -95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO:103.

[0011] The nucleic acid may include or exclude a region that encodes for a self-cleaving peptide or internal ribosome entry sequence (IRES), wherein the self-cleaving peptide or IRES is between the TNFa polypeptide and the TCR or CAR. The self-cleaving polypeptide may comprise the amino acid sequence of SEQ ID NO:2. The self-cleaving polypeptide may comprise the amino acid sequence of SEQ ID NO:5. The self-cleaving polypeptide may comprise an amino acid sequence that is a fragment of SEQ ID NO:2. The self-cleaving polypeptide may comprise an amino acid sequence that is a fragment of SEQ ID NO:5. The self-cleaving polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:2, 5, 89, or 92, or to a fragment of SEQ ID NO:2, 5, 89, or 92. The nucleic acid may comprise or exclude a signal peptide.

[0012] The cell may be one that express a polypeptide encoded by the nucleic acid. The cell(s) may further comprise a nucleic acid encoding a CAR or TCR. The cell may be one that expresses the CAR, TCR, and / or TNFa polypeptide encoded by a heterologous nucleic acid. The cell or cells may comprise a plasmid or viral vector encoding the TNFa polypeptide, wherein the plasmid or viral vector is integrated into the cell’s genome. The CAR or TCR may be expressed from a nucleic acid comprising a constitutive promoter that directs the expression of the CAR or TCR. The CAR or TCR may be expressed from a nucleic acid comprising a conditional promoter that directs the expression of the CAR or TCR.

[0013] The TCR may be an engineered TCR. The TCR may comprise or exclude a single chain TCR. The CAR or TCR may comprise an anti-NY-ESO-1 CAR or an anti-NY-ESO-1 TCR. The CAR or TCR may comprise an anti-GD2 CAR or an anti-GD2 TCR. The CAR or TCR may comprise an anti-HPV CAR or an anti-HPV TCR. The HPV may be HPV-16. The HPV may be HPV-16 E7. The TCR may comprise a TCR-alpha and a TCR-beta polypeptide.

[0014] The TCR-alpha polypeptide may comprise a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 107-109, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 107-109, respectively. The TCR-beta polypeptide may comprise a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 104-106, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 104-106, respectively. Alternatively, the TCRA CDR1 may comprise302139246.1 - 4 -an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 107. The TCRA CDR2 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 108. The TCRA CDR3 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 109. The TCRB CDR1 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 104. The TCRB CDR2 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 105. The TCRB CDR3 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 106.

[0015] The TCR-alpha polypeptide may comprise the amino acid sequence of SEQ ID NO:3. The TCR-alpha polypeptide may comprise an amino acid sequence that is a fragment of SEQ ID NO:3. The The TCR-alpha polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:3 or to a fragment of SEQ ID NO:3. The TCR-beta polypeptide may comprise the amino acid sequence of SEQ ID NO:4. The TCR-beta polypeptide may comprise an amino acid sequence that is a fragment of SEQ ID NO:4. The TCR-beta polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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, 91,302139246.1 - 5 -92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO:4 or to a fragment of SEQ ID NO:4.

[0016] The nucleic acid may comprise the nucleic acid sequence of SEQ ID NO: 72, a fragment of the nucleic acid sequence of SEQ ID NO:72. The nucleic acid may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:72. The nucleic acid may comprise a nucleic acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:72. The nucleic acid may comprise a sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 72 or to a fragment of SEQ IDNO:72.

[0017] The nucleic acid may encode for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide. The TCR-alpha polypeptide may comprise a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 110-112, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:110-112, respectively. The TCR-beta polypeptide may comprise a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS:113-115, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 113-115, respectively. Alternatively, the TCRA CDR1 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 110. The TCRA CDR2 may comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO: 111. The TCRA CDR3 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 112. The TCRB CDR1 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:113. The TCRB CDR2 may comprise an amino acid sequence that has about, or has at least, or has at most, or has exactly 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,302139246.1 - 6 -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 (or any derivable range therein) to SEQ ID NO: 114. The TCRB CDR3 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 115.

[0018] The TCR-alpha polypeptide may comprise the amino acid sequence of SEQ ID NO:91, a fragment of the amino acid sequence of SEQ ID NO:91, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:91, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:91. The TCR-alpha polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:91 or to a fragment of SEQ ID NO:91. The TCR-beta polypeptide may comprise the amino acid sequence of SEQ ID NO:93, a fragment of the amino acid sequence of SEQ ID NO:93, an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 93, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:93. The TCR-beta polypeptide may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:93 or to a fragment of SEQ ID NO:93.

[0019] The nucleic acid may encode an anti-GD2 CAR comprising an anti-GD2 scFv. The anti-GD2 scFv may comprise a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that have the amino acid sequence of SEQ ID NOS:97-102, respectively. Alternatively, the HCDR1 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:97. The HCDR2 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:98. The HCDR3 may comprise an amino acid sequence that has about, or has at least, or has at most, or has exactly 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,302139246.1 - 7 -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 (or any derivable range therein) to SEQ ID NO:99. The LCDR1 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 100. The LCDR2 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 101. The LCDR3 may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO: 102.

[0020] The anti-GD2 scFv may comprise a variable heavy chain region (VH) and variable light chain region (VL) and wherein the VH comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:94. The VH may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:94. The anti-GD2 scFv may comprises a variable heavy chain region (VH) and variable light chain region (VL) and wherein the VL comprises the amino acid sequence of SEQ ID NO:96 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:96. The VL may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:96. The VH may be 3’ proximal to the VL on the nucleic acid. The VL may be 3’ proximal to the VH. The anti-GD2 scFv may comprise the amino acid sequence of SEQ ID NO:86 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:86. The anti-GD2 scFv may comprise an amino acid sequence that has about, or has at least, or has at most, or has 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% sequence identity (or any derivable range therein) to SEQ ID NO:86. A first region is 3’-proximal to a second region when the first region is attached to the 3’ nucleic acid end of the second region. There302139246.1 - 8 -may be further intervening amino acid residues between the first and second regions. Thus, the regions need not be immediately adjacent, unless specifically specified as not having intervening amino acid residues. The term “5 ’-proximal” is similarly defined in that a first region is 5 ’-proximal to a second region when the first region is attached to the 5’ nucleic acid end of the second region. Similarly, there may be further intervening amino acid residues between the first and second regions unless stated otherwise.

[0021] Cell(s) of the disclosure may include or exclude a T cell, a natural killer (NK) cell, a natural killer T cell (NKT), an invariant natural killer T cell (iNKT), stem cell, lymphoid progenitor cell, peripheral blood mononuclear cell (PBMC), bone marrow cell, fetal liver cell, embryonic stem cell, cord blood cell, and / or an induced pluripotent stem cell (iPS cell). The cell may be a T cell or an NK cell. The cell may comprise a naive memory T cell. The naive memory T cell may comprise a CD4+ or CD8+ T cell. The T cell may comprise or exclude a T cell from a population of CD14 depleted, CD25 depleted, and / or CD62L enriched PBMCs.

[0022] Cells include those that have been infected with a virus comprising a nucleic acid of the disclosure. The virus may comprise lentivirus or a lentiviral-derived virus or vector. The cell may be one that is not yet a T cell or NK cell, the method further comprising culturing the cell under conditions that promote the differentiation of the cell into a T cell or an NK cell. The methods may comprise culturing the cell under conditions to expand the cell before and or after introducing the nucleic acid into the cell. The cell may be cultured with serum-free medium. The cell or cells of the disclosure may comprises a nucleic acid encoding a CAR or TCR.

[0023] The population of cells may comprise 103-108cells and include the cells of the disclosure. Any of the cell populations may comprise at least, about, 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, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 103,5 X 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x 105, 2 x 105, 3 x 105, 4 x 105,5 x 105, 6 x 105, 7 x 105, 8 x 105, 9 x 105, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108, 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x io10, 2 x io10, 3 x 1010, 4 x 1010, 5 x 1010, 6 x 1010, 7 x 1010, 8 x 1010, 9 x 1010, 1 x 1011, 2 x 1011, 3 x 1011, 4 x 1011, 5 x 1011, 6 x 1011, 7 x 1011, 8 x 1011, 9 x 1011, 1 x 1012, 2 x 1012, 3 x 1012, 4 x 1012, 5 x 1012, 6 x 1012, 7 x 1012, 8 x 1012, 9 x 1012, 1 x io13, 2 x 1013, 3 x 1013, 4 x 1013, 5 x 1013, 6 x 1013, 7 x 1013, 8 x 1013, 9 x 1013, or 1 x 1Q14cells, or any derivable range therein.302139246.1 - 9 -

[0024] The patient may be one that has and / or has been determined to have a NY-ESO-1+ cancer. The cancer may comprise a NY-ESO-1+ cancer. The cancer may comprise or exclude melanoma or sarcoma. The cancer may comprise or exclude osteosarcoma or neuroblastoma. The cancer may comprise or exclude ovarian or cervical cancer. The cancer may include or exclude a solid tumor. The cancer may include or exclude The cancer may comprise or exclude a cancer described herein. The methods may further comprise administering an additional therapy to the patient. The additional therapy may comprise or exclude an immunotherapy, anti-angiogenic therapy, chemotherapy, surgery, radiotherapy, neoantigen therapy, or vaccination. The additional therapy may comprise or exclude an immunotherapy and wherein the immunotherapy comprises immune checkpoint inhibitor therapy or bispecific T cell engagers. The cells may be autologous or non-autologous. The subject or patient may be a human subject. The subject or patient may be further defined as a mammal, a non-human primate, a pig, horse, mouse, rat, rabbit, or dog. The term “subject” and “patient” is used interchangeably herein.

[0025] When reciting that the CDR1, CDR2, and CDR3 has at least 80% sequence identity to the CDR1, CDR2, and CDR3 or a specific amino acid sequence, one skilled in the art would understand that each of CDR1, CDR2, and CDR3 can share at least 80% identity to CDR, CDR2, and CDR3, respectively of the specific recited amino acid sequences.

[0026] The TNFa-armored cells of the disclosure may be useful for treating subjects for cancer. Surprisingly, it was found that the TNFa-armored cells with a TCR or CAR did not exhibit any antigen-independent translation or secretion. The cells of the disclosure may also exhibit a level of toxicity that is less than expected, given the finding that there is no exhibition of antigen-independent translation or secretion.

[0027] The therapeutic agents of the disclosure may be used for in vivo, in vitro, or ex vivo administration. The route of administration of the therapies may be, for example, intracutaneous, subcutaneous, intravenous, local, topical, and intraperitoneal administrations.

[0028] In some embodiments, the disclosed methods are directed to methods for treating cancer. The cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may be recurrent, metastatic, relapsed, or of a Stage I, II, III, or IV.

[0029] The references to the methods of treatment by therapy or surgery or in vivo diagnosis methods in example 1 of this description and in the claims and disclosure of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods.302139246.1 - 10 -

[0030] The compositions, methods, nucleic acids, and the cells of the disclosure may be useful in the treatment of diseases and may have advantageous effects such as, reducing tumor burden; sustaining potent antitumor activity within the suppressive tumor environment without causing systemic toxicity; achieving markedly improved tumor control across multiple solid tumor models (melanoma, cervical carcinoma, and osteosarcoma) without inducing systemic inflammation or end-organ damage; restricting the increased TNF-a activity to antigen-positive tumors; producing no significant differences in either inflammatory tissue damage / lymphocytic infiltration of colons, or elevations in stool samples from subjects treated with TNF-a-armed TCR / CAR-T cells when compared to the corresponding conventional TCR / CAR; producing no significant increases in autoimmune activity in treated subjects or in the organs of treated subjects; reducing Treg proportions in subjects; reducing serum IL- 10 levels in subjects; reducing serum Th-2 associated cytokines; reducing serum IL-4 and IL-5; increasing CD4 naive and CD8 effector populations; increasing CD4 and CD8 central memory populations; and / or reducing T cell exhaustion. One surprising effect is the increase in the TNF-a translation and secretion upon antigen stimulation of the TCR / CAR-T cell.

[0031] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0032] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0033] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0034] 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.

[0035] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The302139246.1 - 11 -phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0036] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0037] 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 the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] 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.

[0039] FIG. 1A-1E shows that TNF-alpha cytokine functionality was associated with a superior clinical response to transgenic TCR-T cell therapy, while CD4 Th2 cytokine functionality was associated with an inferior clinical response. FIG. 1A shows a schematic of the multiplex single-cell procedure used to assess the cytokine secretion profile of clinical anti-NY-ESO-1 TCR-T cells that were used to treat patients with melanoma or sarcoma (n=15). Pre-infusion cell products were co-cultured with K562 target cells transduced with HLA-A:02:01 and pulsed with corresponding NY-ESO-1 peptide. Stimulated cells were analyzed on an IsoPlexis single-cell barcode chip system. FIG. IB shows that the CD8 TNF-alpha302139246.1 - 12 -functional strength index (FSI), i.e., the percentage of single-cells secreting TNF-alpha multiplied by the degree of cytokine secretion intensity, was significantly greater in clinical responders to therapy vs non-responders. FIG. 1C shows that CD8 TNF-alpha FSI was correlated with a superior degree of anti-tumor response (peak percent change in tumor burden per RECIST1.1 criteria). FIG. ID shows that CD4 Th2 polyfunctional strength index (PSI, i.e., the proportion of single cells secreting Th2 cytokines IL-4, IL-5, IL-10, and IL-13 multiplied by the signal intensity of those cytokines) was significantly greater in clinical non-responders to therapy vs responders. FIG. IE shows thatNY-ESO-1 TCR-T cells cultured in the presence of recombinant TNF-alpha (50 ng / mL) for 72 hours and then stimulated with their target antigen were found to display significantly lower CD4 Th2 PSI cytokinetic functionality compared to control TCR-T cells. *p<0.05, **p<0.01, unpaired t-test.

[0040] FIG. 2 shows a lentiviral vector map of NY-ESO-1 TCR co-packaged with TNF-alpha. A novel lentiviral vector was designed that includes the affinity-enhanced NY-ESO-1 TCR alpha and beta chains, along with TNF-alpha (~lkb). This was termed a TNF-alpha-“armed” TCR vector. Transduction efficiency was enhanced by incorporating LentiBOOST®, a universal poloxamer-based, receptor-independent adjuvant which facilitates fusion of lentivirus particles with cell membranes. As the TCR is HLA-A:02:01, it is critical that tumor cells express both the target antigen and possess the correct HLA-A type for the TCR to engage with the target.

[0041] FIG. 3A-3D shows that transduction with a TNF-alpha-armed NY-ESO-1 TCR vector resulted in increased expression of TNF-alpha, as well as increased antigen-dependent secretion of TNF-alpha. FIG. 3A shows representative flow cytometry data showing surface expression of NY-ESO-1 TCR via MHC dextramer binding in cells transduced with the lentiviral vector encoding the NY-ESO-1 TCR alone (pRRL-NYESOlTCR) or the armed vector (pRRL-NYESOlTCR-TNFA). Gating quantities represent the percentage of TCR+cells within the CD3+population. FIG. 3B shows qRT-PCR results that showed increased expression of TNF-alpha in T-cells transduced with the armed vector (pRRL-NYESOlTCR-TNFA) compared to the cells transduced with the lentiviral vector encoding the NY-ESO-1 TCR alone (pRRL-NYESOlTCR) or mock transduced T-cells (fold increase compared to calibrator gene SDC4). Values represent the mean of three separate experiments, + / - standard deviation. FIGS. 3C-D show multiplexed sandwich ELISA results comparing secretion levels of several effector cytokines in human T-cells transduced with the TNF-alpha armed TCR lentiviral vector vs those transduced with the vector only encoding the TCR. Despite the increased expression of TNF-alpha seen in FIG. 3B, TNF-alpha armed T-cells did not show a302139246.1 - 13 -significant increase in the secretion of effector cytokines without antigen stimulation (FIG.3C). However, when stimulated with K562, HLA-A:02:01+cells, pulsed with the appropriate NY-ESO-1 peptide, the TNF-alpha secretion levels from the T-cells transduced with the TNF-alpha-armed TCR vector were significantly greater (~4-fold) than the control cells transduced with the TCR alone (FIG. 3D). Values represent the mean of three separate experiments, + / -standard deviation. * p<0.05, *** p <0.001, unpaired t-test.

[0042] FIG. 4A-4B shows that transduction with a TNF-alpha-armed NY-ESO-1 TCR vector resulted in superior target cell killing in vitro. FIG. 4A shows T-cell killing of M257-A2 melanoma cells, which are HLA-A:02:01 -positive, express NY-ESO-1, and were engineered to express nuclear red fluorescent protein (nRFP). Tumor cells were co-cultured with T-cells transduced with lentiviral vectors encoding the NY-ESO-1 TCR alone (pRRL-NYESO1TCR), or with an armed lentiviral vector encoding both TNF-alpha and the NY-ESO-1 TCR (pRRL-NYESOlTCR-TNFA). Culture conditions were at an effectortarget ratio of 2:1, and were plated in triplicates. Cell proliferation and cell death (reported as confluency of culture) were measured by nRFP real time imaging using an IncuCyte® ZOOM (Essen) for 48 hours. Each timepoint represents the average fluorescence confluency + / - standard deviation.FIG. 4B shows that the TNF-alpha-armed TCR T-cells yielded significantly improved cell killing activity, as measured by a greater decrease in nRFP cell confluency at the conclusion of the assay. *** p<0.001, unpaired t-test.

[0043] FIG. 5A-5B shows an overview of an in vivo xenograft model of NY-ESO-1 TCR-T cell therapy. FIG. 5A shows that T-cells from commercially available human donors can be transduced with lentiviral vectors containing a mock TCR, NY-ESO-1 TCR, or the TNF-alpha-armed NY-ESO-1 TCR vector. Following cell expansion and lot release testing, cells can be frozen. In parallel, NSG mice, non-obese diabetic (NOD) / severe combined immunodeficiency (SCID) / / / 2 / g mice can be implanted with A375 melanoma cells which express NY-ESO-1 and are HLA-A:02:01 -positive. Seven days after tumor implantation, T-cells can be transferred into the tumor-bearing mice. FIG. 5B shows representative data of this system comparing control (mock) T-cells and NY-ESO-1 TCR transduced T-cells. **** p<0.0001, unpaired t-test, n = four mice per group.

[0044] FIG. 6A-6B shows results from GeoMx spatial profiling revealing that T-cell infiltration patterns were anti-correlated with WNT / beta-catenin expression. FIG. 6A shows confocal microscopy images from the GeoMx platform overlaying nuclear staining (blue), NY-ESO-1 (yellow), pan-Cytokeratin (green), and CD45 (red). The images were used to identify regions-of-interest (ROIs; indicated by circles) in primary, metastatic, and recurrence samples302139246.1 - 14 -from a patient with sarcoma who was treated with transgenic NY-ESO-1 TCR-T-cell immunotherapy. Pink circles in the metastasis lesion indicate those that were within the tumor region. FIG. 6B shows raw expression of beta-catenin (CTNNB1), CD8 T-cells (CD8A), and CD4 T-cells (CD4) on the y-axis across ROIs derived from each sample (denoted by color), which demonstrated that beta-catenin expression is associated with significant CD8 T-cell exclusion in metastatic and recurrence samples, and CD4 T-cell exclusion in metastatic samples *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, pairwise Wilcox test.

[0045] FIG. 7A-7B shows that TNF-alpha treatment of M257-A2 melanoma cells caused cell death associated with p-STATl and p-JNK signaling pathways. FIG. 7A shows mass cytometry results of phospho-signaling proteins in M257-A2 cell lines, which express NY-ESO-1 and HLA-A:02:01. Cells were treated with TNF-alpha (1000 lU / mL) or vehicle (control) for 48 hours. TNF-alpha-treated cells exhibiting cell death associated with increases in p-STATl and p-JNK signaling. Cells were read on a Helios Mass Cytometer and analyzed using Cytofkit, where fold change (FC) with respect to time 0 was calculated for median intensity of each protein. FIG. 7B shows that 48-hour fold change levels of p-STATl and p-JNK were significantly greater in cells treated with TNF-alpha compared to control cells. *p<0.05; **p<0.01, unpaired t-test.

[0046] FIG. 8A-8C shows that CD4 Th2 cytokine polyfunctionality was associated with an inferior clinical response, and TNF-alpha suppressed Th2 polyfunctionality and augmented Th9 polyfunctionality. FIG. 8A shows that CD4 Th2 polyfunctional strength index (PSI, i.e., proportion of single cells secreting the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 multiplied by the signal intensity of those cytokines) was significantly greater in clinical non-responders to therapy vs responders in a cohort of patients with melanoma and sarcoma who were treated with transgenic NY-ESO-1 TCR T-cell therapy (n=15). FIGS. 8B-C show that NY-ESO-1 TCR-T cells cultured in the presence of recombinant TNF-alpha (50 ng / mL) for 72 hours and then stimulated with their target antigen displayed significantly lower CD4 Th2 PSI cytokinetic functionality compared to control TCR-T cells, while their CD4 Th9 PSI (i.e., the proportion of cells secreting IL-9 multiplied by degree of cytokine intensity) was significantly greater compared to control cells. Data in FIGS. 8B-C represent three experimental replicates, *p<0.05, ***p<0.001, unpaired t-test.

[0047] FIG. 9 shows an overview of repetitive tumor stimulation assays.

[0048] FIG. 10A-10B shows that TNF-alpha-armed NY-ESO-1 TCR transgenic T-cells generated greater TNF-alpha secretion levels in tumor cell co-culture media over time. FIG.10A shows results from T-cells transduced with the lentiviral vector encoding the NY-ESO-1302139246.1 - 15 -TCR alone (pRRL-NYESOlTCR) or the armed vector (pRRL-NYESOlTCR-TNFA) that were serially co-cultured with NY-ESO-1+, HLA-A:02:01+, M407 melanoma cells in triplicates, as described above, and their culture media analyzed for TNF-alpha levels at the end of each 48-hour co-culture via ELISA. FIG. 10B shows that the area under the curve (AUC) for TNF-alpha levels was significantly greater (~2-fold) for the armed TCR vector compared to the control vector (* p<0.05, unpaired t-test).

[0049] FIG. 11 shows an overview of the IsoPlexis single-cell intracellular phospho-proteome barcode chip assay. This enables the simultaneous interrogation of up to 15 phosphoproteins involved in various cell signaling pathways.

[0050] FIG. 12 shows that CD4 Th2 cytokine polyfunctionality is associated with increased signaling via NF-kB pathway. CD4 Th2 polyfunctional strength index (PSI, i.e., proportion of single cells secreting the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 multiplied by the signal intensity of those cytokines) from a clinical cohort of NY-ESO-1 TCR-T infusion products was found to be directly proportional to phospho-IkB A functional strength index (FSI, i.e., proportion of single cells from the same sample which were positive for p-IkBA multiplied by the signal intensity of that phospho-protein). This was consistent with previous work associating increased NF-kB signaling activity in the setting of reduced TNF-alpha / TNFR2 signaling as promoting CD4 Th2 polarization.

[0051] FIG. 13 shows validation of TNFR2 silencing in human peripheral mononuclear blood cells (PBMCs). qRT-PCR revealed near-complete loss of TNFR2 expression in human PBMCs following CRISPR knockdown of TNFR2 (fold change compared to calibrator gene SDC4). Values represent the mean of three separate experiments, + / - standard deviation.

[0052] FIG. 14A-14F. Lentiviral Vector maps that include (A) 1GY NY-ESO-1 TCR; (B) HPV16 E7 TCR; (C) GD2 GAR-28z; (D) TNF-a-armed 1GY NY-ESO-1 TCR; (E) TNF-a-armed HPV16 E7 TCR; and (F) TNF-a-armed GD2 CAR-28z.

[0053] FIG. 15A-15L TNF-a-armed TCR / CAR-T cells possess increased basal expression of TNF-a mRNA, as well as increased antigen-dependent secretion of TNF-a protein and tumor cell killing in vitro. qRT-PCR reveals increased expression of TNF-a mRNA in T-cells transduced with the TNF-a-armed variants of the NY-ESO-1 TCR (A), the HPV16 E7 TCR (B), or GD2 CAR (C) compared to both the cells transduced with the lentiviral vector encoding the conventional TCR / CAR alone and mock transduced T-cells (fold increase compared to calibrator gene SDC4). Values are the mean of three separate experiments, + / -standard deviation. TNF-a-armed TCR / CAR-T cells display significantly increased secretion302139246.1 - 16 -levels TNF-a when co-cultured with corresponding antigen-positive tumor cells for 20 hours (D, NY-ESO-1 TCR vs NY-ESO-1+, HLA-A*02:01+ A375 melanoma cells; E, HPV16 E7 TCR vs HPV16E7+, HLA-A*02:01+ CaSki cervical carcinoma cells, F, GD2 CAR vs GD2+ 143B osteosarcoma cells), but display no significant differences in antigen-dependent IFN-y secretion, and no significant differences in basal (unstimulated) secretion of TNF-a or IFN-y in any of the constructs tested. * p<0.05, ** p <0.01, **** pO.OOOl, unpaired t-test. Comparison of cancer cell killing activity under chronic antigen stimulation conditions in vitro as measured by real time fluorescence imaging during first and fifth stimulation with co-culture of A375 cells with TNF-a-armed and conventional NY-ESO-1 TCR-T cells (G), 4050 cells with TNF-a-armed and conventional HPV16 E7 TCR-T cells (I), and 143B cells with TNF-a-armed and conventional GD2 CAR-T cells (K), with corresponding TNF-a concentrations in media and the end of each stimulation (H, J, L). Dashed lines for datasets represent normalized initial cancer cell concentration and TNF-a lower limit of detection, respectively. ** p<0.01, **p<0.01, ****p<0.0001, two-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test. Data reported as mean + / - standard deviation.

[0054] FIG. 16A-16F. Treatment with TNF-a-armed TCR / CAR-T cells results in superior tumor control and survival in vivo. (A) NSG mice were administered IxlO6A375 melanoma cells via flank injection. Seven days after tumor seeding, tumor-bearing mice were injected with 3xl06mock TCR-T cells, conventional NY-ESO-1 TCR-T cells, or TNF-a-armed NY-ESO-1 TCR-T cells, and tumor growth was monitored via caliper measurements, *** p<0.001, ****p<0.0001, two-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test. Data reported as mean + / - standard deviation from n=9 mice per group. (B) TNF-a-armed NY-ESO-1 TCR-T cell therapy results in superior tumor-bearing mice survival over conventional NY-ESO-1 TCR-T cells or mock TCR-T cells, **p<0.01, log-rank (Mantel-Cox) test. (C) NSG mice were administered IxlO64050 cervical carcinoma cells via flank injection. Seven days after tumor seeding, tumor-bearing mice were injected with 3xl06mock TCR-T cells, conventional HPV16 E7 TCR-T cells, or TNF-a-armed HPV16 E7 TCR-T cells, and tumor growth was monitored via caliper measurements, ** p<0.01, ****p<0.0001, two-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test. Data reported as mean + / - standard deviation from n=9 mice per group. (D) TNF-a-armed HPV16 E7 TCR-T cell therapy results in superior tumor-bearing mice survival over conventional HPV16 E7 TCR-T cells or mock TCR-T cells, **p<0.01, log-rank (Mantel-Cox) test. (E) NSG302139246.1 - 17 -mice were administered IxlO6143B osteosarcoma cells via flank injection. Seven days after tumor seeding, tumor-bearing mice were injected with 3xl06mock CAR-T cells, conventional GD2 CAR-T cells, or TNF-a-armed GD2 CAR-T cells, and tumor growth was monitored via caliper measurements, * p<0.05, two-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test. Data reported as mean + / - standard deviation from n=7 mice per group. (F) TNF-a-armed GD2-T cell therapy results in superior tumor-bearing mice survival over conventional GD2 CAR-T cells alone or mock CAR-T cells, *p<0.01, log-rank (Mantel-Cox) test.

[0055] FIG. 17A-17D Lack of inflammatory colitis during in vivo treatment with TNF-a-armed TCR / CAR-T cells. (A) Comparison of intestinal inflammation assessment on histology among treatment groups. Severity score is the sum of intestinal inflammation scored for inflammatory cell infiltrate, epithelial cell changes, and mucosal architecture as previously reported (28). Comparison by Kruskal-Wallis test with a significance threshold of 0.05. Stool calprotectin levels were also measured weekly in all mouse cohorts throughout the course of treatment (B), NY-ESO-1 TCR system, (C), HPV16 E7 TCR system, (D), GD2 CAR system) and were compared via two-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test; no significant differences between groups were found.

[0056] FIG. 18A-18F. Treatment with TNF-a-armed TCR / CAR-T cells is associated with alterations in T-cell phenotypes and associated cytokines in peripheral circulation. (A) Peripheral proportions of CD4 Tregs and Th2 cells in mice treated with TNF-a-armed NY-ESO-1 TCR-T cells compared to conventional NY-ESO-1 TCR-T cells, along with serum concentrations of their associated cytokines IL-10, IL-4, and IL-5 obtained at day +14 (B). (C) Peripheral proportions of CD4 Tregs, CD4 Th2 cells, CD4 naive cells, and CD8 effector cells in mice treated with TNF-a-armed HPV16 E7 TCR-T cells compared to conventional HPV16 E7 TCR-T cells. (D) Serum levels of cytokines IL-10, IL-4, and IL-5 obtained at day +14 from the HPV16 E7 TCR cohort (E) Peripheral proportions of CD4 Tregs in mice treated with TNF-a-armed GD2 CAR-T cells, along with serum concentrations of their associated cytokines IL-10, IL-4, and IL-5 obtained at day +14 (F). * p<0.05, **p<0.01, ***p<0.001, unpaired t-test.

[0057] FIG. 19A-19D Alterations in intratumoral T-cell infiltration patterns within the tumor microenvironment of mice treated with TNF-a-armed TCR / CAR-T cells. (A) Quantitative immunohistochemistry analysis of human CD3+ cell infiltration / mm2in tumors from mice treated with TNF-a-armed TCR / CAR-T cells compared to their corresponding conventional TCR / CAR. (B) Intratumoral porportions of CD4 Tregs, CD4 Th2, CD4 naive,302139246.1 - 18 -and CD4 effector phenotype cells in mice treated with the TNF-a-armed HPV16 E7 TCR-T cells compared to the conventional HPV16 E7 TCR-T cells. (C) Intratumoral porportions of CD4 Tregs, CD4 Thl7, CD4 central memory, and CD8 central memory phenotype cells in mice treated with the TNF-a-armed GD2 CAR-T cells compared to the conventional GD2 CAR-T cells. (D) Intratumoral proportions of G-MDSCs and M-MDSCs in mice treated with TNF-a-armed GD2 CAR-T cells compared to the conventional GD2 CAR-T cells and the mock transduced T-cells. * p<0.05, ** p<0.01, *** p<0.0001, ****p<0.0001, unpaired t-test.

[0058] FIG. 20A-20H. Spatial transcriptomic analyses of tumor microenvironment in mice treated with TNF-a-armed TCR / CAR-T cells. (A) Relative enrichment of TNF-a signaling in tumor cells via p38 and NF-KB pathways. (B) Relative enrichment of TNF-a signaling in T-cells via NF-KB pathways. (C) Relative enrichment of VEGFA / TGFB+ signaling clusters in tumor cells. (D) Extrapolative clustering of tumor cell signaling pathways, demonstrating relative abundance and signaling intensity of component genes. (E,F) T-cell phenotype transcriptional signature relative enrichment in tumor infiltrating T-cells comparing effector memory phenotype vs naive and memory compartments (E) and naive vs memory and central memory phenotypes (F). (G) Relative enrichment of effector T-cell signatures in tumor infiltrating T-cells. (H) Relative enrichment of PDl-low vs PDl-high transcriptional signatures in tumor infiltrating T-cells. Statistical comparisons for transcriptional signaling clusters in all models aggregated by treatment type (TNF-a-armed vs conventional TCR / CAR-T cells) and compared by Mann-Whitney U test, with p values noted in the y-axis of each graph, with significance set at p<0.1.

[0059] FIG. 21A-21I. Cumulative temperature, body weight, and body condition scores from in vivo testing of TNF-a-armed TCR / CAR-T cell treatment. Mouse temperature (A), body weight % change from baseline obtained on day of adoptive cell transfer (B), and body condition score (C) of NSG mice treated with mock TCR, conventional NY-ESO-1 TCR, or TNF-a-armed NY-ESO-1 TCR-T cells. (D, E, F) Corresponding temperature, body weight, and body condition scores, respectively, for the HPV16 E7 TCR-T cell in vivo experiments. (G, H, I) Corresponding temperature, body weight, and body condition scores, respectively, for the GD2 CAR-T cell in vivo experiments. Data were compared via two-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test; no significant differences between groups were found.

[0060] FIG. 22A-22C. No difference in longitudinal serum concentrations of TNF-a or IL-6 in mice treated with TNF-a-armed TCR / CAR-T cells. Day +7 and Day +14302139246.1 - 19 -concentrations of TNF-a and IL-6 from mice treated with mock, conventional, or TNF-a-armed NY-ESO-1 TCR-T cells (A), HPV16 E7 TCR-T cells (B), or GD2 CAR-T cells (C). No significant differences were observed. Comparison by Kruskal-Wallis test with a significance threshold of 0.05.

[0061] FIG. 23A-23C. Lack of increased end-organ immune infiltration during treatment with TNF-a-armed TCR / CAR-T cells in vivo. Summary of immune assessment histopathological scoring for all organs assessed at time of sacrifice for the NY-ESO-1 TCR system (A), the HPV16 E7 TCR system (B), and the GD2 system (C). Comparison by Kruskal-Wallis test with a significance threshold of 0.05.DETAILED DESCRIPTION OF THE INVENTIONI. T Cell Receptor (TCR) and Methods for Generating Engineered TCRs.

[0062] The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (T cells) that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains (that is, it is a heterodimer). In 95% of T cells in humans, the TCR consists of an alpha (a; also referred to herein as “a”) and beta (P - also referred to herein is “b”) chain, whereas in 5% of T cells the TCR consists of gamma and delta (y / 5) chains. This ratio changes during ontogeny and in diseased states as well as in different species.

[0063] When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors. The TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (P) chains expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as a:P (or aP or ab) T cells, though a minority of T cells express an alternate receptor, formed by variable gamma (y - also referred to herein as “g”) and delta (8 -also referred to herein as “d”) chains, referred as y6 (or gd) T cells.

[0064] Each chain is composed of two extracellular domains: Variable (V) region and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel P-sheets. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide / MHC complex.302139246.1 - 20 -

[0065] The variable domain of both the TCR a-chain and P-chain each have three hypervariable or complementarity determining regions (CDRs), whereas the variable region of the P-chain has an additional area of hypervariability (HV4) that does not normally contact antigen and, therefore, is not considered a CDR.

[0066] The residues are located in two regions of the TCR, at the interface of the a- and P-chains and in the P-chain framework region that is thought to be in proximity to the CD3 signaltransduction complex. CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the P-chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC. CDR4 of the P-chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which forms a link between the two chains.

[0067] The TCR being a member of the IgSF protein means it may be compared to antibodies and BCR. In terms of similarity, TCR is like half an antibody with a heavy and a light chain, except the heavy chain is without its crystallisable fraction (Fc) (Note: ontogenically TCR alpha undergo VJ recombination, so it is like a light chain; TCR beta undergoes VDJ recombination, so it is like a heavy chain). So the TCR is ontologically like one of the antibody-binding fragments of the antibody. The two subunits of TCR are twisted together. Whereas the antibody uses its Fc region to bind to Fc Receptors on innate leukocytes, TCR is already docked onto the cell membrane. However, it is not able to mediate signal transduction itself due to its short cytoplasmic tail, so TCR still requires CD3 and zeta to carry out the signal transduction in its place, just as antibodies requires binding to FcRs to initiate signal transduction. In this way the MHC-TCR-CD3 interaction for T cells is functionally similar to the Ag-Ig-FcR interaction for myeloid leukocytes, and Ag-Ig-CD79 interaction for B cells.

[0068] The exogenous TCR may comprise proteins expressed from TCR-alpha and TCR-beta genes. The exogenous TCR may comprise proteins expressed from TCR-gamma and TCR-delta genes. The exogenous TCR may comprise proteins expressed from TCR-alpha and TCR-beta genes and the antigen recognition receptor comprises proteins expressed from the TCR-gamma and TCR-delta genes. The exogenous TCR may comprise proteins expressed from TCR-gamma and TCR-delta genes and the antigen recognition receptor comprises proteins expressed from the TCR-alpha and TCR-beta genes.302139246.1 - 21 -

[0069] Methods of generating antigen-specific TCRs are known in the art. Methods may include, for example, 1) Synthesizing known or predicted HLA-restricted peptide epitopes derived from proteins of interest (e.g. tumor antigens, neoantigens from sequencing data, efc.); 2) presenting these via an antigen-presenting cell (for expansion) or tetramer (for direct sorting) to a pool of T cells from which TCR sequences are to be extracted (e.g. tumor infiltrating lymphocytes in the case of tumor-ag specific T cells); 3) selecting or screening for antigenspecific T cells (e.g. FACS sorting antigen-specific T cells based on tetramer binding); 4) cloning (via RT-PCR) and sequencing the TCR genes (i.e. alpha and beta chains or gamma and delta chains of the TCRs); cloning and sequencing may be done either on a population or single cell level; and 5) confirming and analyzing TCR specificity by, for example, testing the function of TCR clones by transducing peripheral blood T cells with these sequences and assessing their reactivity to target cells that express the cognate peptide-MHC complex. Reactivity is usually measured based on cytokine production (e.g. interferon gamma).II. Chimeric Antigen Receptors (CARs) and Methods of Generating CARs.

[0070] The term “chimeric antigen receptor” or “CAR” refers to engineered receptors, which graft an arbitrary specificity onto an immune effector cell. These receptors are used to graft the specificity of a monoclonal antibody onto an immune cell; with transfer of their coding sequence facilitated by retroviral or lentiviral vectors. The receptors are called chimeric because they are composed of parts from different sources.

[0071] The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain, CD28 or 4 IBB intracellular domains, or combinations thereof. Such molecules result in the transmission of a signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When immune cells express this molecule (as an example achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g. neuroblastoma cells).

[0072] The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original302139246.1 - 22 -molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.

[0073] The CARs may comprise at least one extracellular and at least one intracellular domain. An extracellular domain can comprise a target-specific binding element otherwise referred to as an antigen- or ligand-binding moiety that specifically binds to any particular antigen of interest.

[0074] The intracellular domain or the cytoplasmic domain may comprise, one or more costimulatory signaling region(s), and / or a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules may be cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of immune cells to antigen.A. Signal Peptide

[0075] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. A signal peptide may direct the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g. in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0076] The signal peptide may be cleaved after passage of the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. A restriction site may be at the carboxy end of the signal peptide to facilitate cleavage.B. Antigen Binding Domain

[0077] Polypeptides of the present disclosure may comprise one or more antigen binding domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. An antigen binding domain may be a single-chain variable fragment (scFv) based on one or more antibodies (e.g., CD20 antibodies). An antigen binding domain may comprise a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide. The antigen binding domain may302139246.1 - 23 -comprise a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0078] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0079] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0080] It is also contemplated that the antigen binding domain may be multi-specific or multivalent by multimerizing the antigen binding domain with VH and VL region pairs that bind either the same antigen (multi -valent) or a different antigen (multi-specific).

[0081] The binding affinity of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, 10'10M, 10-11M, 10'12M, or 10'13M, or any derivable range therein. The KD of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, 10'10M, 10-11M, 10'12M, or 10'13M (or any derivable range therein).

[0082] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0083] The polypeptide comprising the humanized binding region may have equal, better, or at least 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, 104, 106, 106, 108, 109, 110, 115, or 120% (or any derivable range therein)302139246.1 - 24 -binding affinity and / or expression level in host cells, compared to a polypeptide comprising a non-humanized binding region, such as a binding region from a mouse.

[0084] The framework regions, such as FR1, FR2, FR3, and / or FR4 of a human framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a mouse framework.

[0085] The framework regions, such as FR1, FR2, FR3, and / or FR4 of a mouse framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, contiguous amino acid additions, or contiguous amino acid deletions with respect to a human framework.

[0086] The substitution may be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of FR1, FR2, FR3, or FR4 of a heavy or light chain variable region.302139246.1 - 25 -C. Extracellular Peptide Spacer

[0087] Between the extracellular domain and the transmembrane domain of the CAR, and / or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. An extracellular spacer may link the antigen-binding domain to the transmembrane domain. A peptide spacer may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding.

[0088] The spacer may comprise the hinge region from IgG. The spacer may comprise or further comprise the CH2CH3 region of immunoglobulin and portions of CD3. The CH2CH3 region may have L235E / N297Q or L235D / N297Q modifications, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity of the CH2CH3 region. The spacer may be from IgG4. An extracellular spacer may comprise a hinge region.

[0089] As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region” or “spacer”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” or “spacer” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl, for example (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No.5,677,425. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions. Other alternatives include the CH2CH3 region of immunoglobulin and portions of CD3.

[0090] The extracellular spacer can have a length of at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,302139246.1 - 26 -237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any derivable range therein). The extracellular spacer may consist of or comprise a hinge region from an immunoglobulin (e.g. IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.

[0091] The length of an extracellular spacer may have effects on the CAR’s signaling activity and / or the CAR-T cells’ expansion properties in response to antigen-stimulated CAR signaling. A shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. A longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 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, 260, 270, 280, or 290 amino acids may have the advantage of increased expansion in vivo or in vitro.

[0092] As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences:Table: Exemplary Hinge Regions

[0093] The extracellular spacer can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4 hinge region. The extracellular spacer may also include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-302139246.1 - 27 -occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 17).

[0094] The extracellular spacer can comprise an amino acid sequence derived from human CD8; e.g., the hinge region can comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18), or a variant thereof.

[0095] The extracellular spacer may comprise or further comprise a CH2 region. An exemplary CH2 region is APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 19). The extracellular spacer may comprise or further comprise a CH3 region. An exemplary CH3 region is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:20).

[0096] When the extracellular spacer comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 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, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. The extracellular spacer may consist essentially of a hinge, CH2, and / or CH3 region, meaning that the hinge, CH2, and / or CH3 region is the only identifiable region present and all other domains or regions are excluded, but further amino acids not part of an identifiable region may be present.D. Transmembrane Domain

[0097] With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. A transmembrane domain may be a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0098] The transmembrane domain that naturally is associated with one of the domains in the CAR may be used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane302139246.1 - 28 -domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0099] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Illustrative, but non-limiting, examples of transmembrane regions of particular use in the CAR constructs contemplated here can be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain can be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine. A triplet of phenylalanine, tryptophan and valine may be be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, e.g., between 2 and about 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet may provide a particularly suitable linker.

[0100] The transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. The transmembrane domain may be interposed between the extracellular spacer and one or more costimulatory regions. A linker may be between the transmembrane domain and the one or more costimulatory regions.

[0101] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. The transmembrane domain may be derived from CD28, CD8, CD4, CD3-zeta, CD134, or CD7.

[0102] Exemplary transmembrane domains useful in any of the aspects of the disclosure include those in the table below:Table: Exemplary transmembrane domain sequences302139246.1 - 29 -E. Cytoplasmic Domain

[0103] The cytoplasmic domain or otherwise the intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. After antigen and / or ligand recognition, receptors cluster and a signal is transmitted to the cell through the cytoplasmic region. The cytoplasmic region may comprise an intracellular signaling domain. An intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains. The costimulatory domains described herein may be part of the cytoplasmic region.

[0104] The term “effector function” refers to a specialized function of a cell. An effector function of a T cell, for example, may be cytolytic activity, or helper activity including the secretion of cytokines. Thus the term “intracellular signaling domain” refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0105] Cytoplasmic regions and / or costimulatory regions suitable for use in the CARs of the disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g. , cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen binding domain. The cytoplasmic region may comprise at least one (e.g., one, two, three, four, five, six, etc.) IT AM motif as described herein. The cytoplasmic region may comprise DAP10 / CD28 type signaling chains. The cytoplasmic region may comprise CD3-zeta, DAP10, CD28, 2B4,302139246.1 - 30 -DNAM-1, 4-1BB, 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, and NKG2C type signaling chains.

[0106] Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An IT AM motif is YX1X2(L / I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L / I))(X3)n(YXlX2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0107] A suitable cytoplasmic region may be an LT AM motif-containing a portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0108] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that may be incorporated in a CAR of the disclosure:

[0109] The cytoplasmic region may be derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DN AX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase- binding protein; killer activating receptor associated protein; killer- activating receptor- associated protein; etc.}. A suitable cytoplasmic region can comprise an ITAM motif-containing a portion of the full length DAP12 amino acid sequence.

[0110] The cytoplasmic region may be derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc. . A suitable cytoplasmic region may comprise an ITAM motif-containing a portion of the full length FCERI G amino acid sequence.[OHl] The cytoplasmic region may be derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta;302139246.1 - 31 -CD3d antigen, delta polypeptide (TiT3 complex); 0KT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). A suitable cytoplasmic region can comprise an ITAM motif-containing a portion of the full length CD3 delta amino acid sequence.

[0112] The cytoplasmic region may be derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, efc.). A suitable cytoplasmic region can comprise an ITAM motif-containing a portion of the full length CD3 epsilon amino acid sequence.

[0113] The cytoplasmic region may be derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3 -GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). A suitable cytoplasmic region can comprise an ITAM motif-containing a portion of the full length CD3 gamma amino acid sequence.

[0114] The cytoplasmic region may be derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, efc.). A suitable cytoplasmic region can comprise an ITAM motif-containing a portion of the full length CD3 zeta amino acid sequence.

[0115] The cytoplasmic region may be derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane- bound immunoglobulin-associated protein; surface IgM-associated protein; efc.). A suitable cytoplasmic region can comprise an ITAM motif-containing a portion of the full length CD79A amino acid sequence.

[0116] Suitable cytoplasmic regions can comprise a CD28 type signaling chain. Further cytoplasmic regions suitable for use in the CARs of the disclosure include a ZAP70 polypeptide.

[0117] Specific exemplary cytoplasmic regions are known in the art and further shown in the table below.Table: Cytoplasmic Regions302139246.1 - 32 -302139246.1 - 33 -F. Co-Stimulatory Region

[0118] The term “co- stimulatory ligand,” as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule or domain on an immune effector cell, thereby302139246.1 - 34 -providing a signal which, in addition to the primary signal to mediate the immune effector cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on an immune effector cell. A “co-stimulatory molecule” refers to the cognate binding partner on an immune effector cell that specifically binds with a co-stimulatory ligand, thereby mediating a co- stimulatory response by the immune effector, such as, but not limited to, proliferation and / or activation. A “co-stimulatory signal”, as used herein, refers to a signal that in combination with a primary signal, leads to immune cell activation, proliferation, and / or upregulation or downregulation of key molecules.

[0119] By the term “stimulation,” it is meant a primary response induced by binding of a stimulatory molecule with its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction. Stimulation can mediate altered expression of certain molecules. A “stimulatory molecule,” as the term is used herein, means a molecule on an immune effector cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on an immune effector cell, thereby mediating a primary response by the immune effector cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.

[0120] Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, CD40, GITR, 2B4, DNAM-1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and HVEM.

[0121] A co- stimulatory region may have a length of at least, at most, or exactly 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein.

[0122] The costimulatory region may be derived from DAP10 (also known as HCST, DAP 10, KAP10, PIK3AP, hematopoietic cell signal transducer; etc.). The costimulatory region may be derived from an intracellular portion of the transmembrane protein 4- IBB (also known as Tumor necrosis factor receptor superfamily member 9, TNFRSF9; CD137; CDwl37; ILA; etc.). The costimulatory region may be derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). The costimulatory region may be derived from an intracellular portion of the transmembrane protein ICOS (also known as inducible T-302139246.1 - 35 -cell costimulatory, AILIM, CD278, and CVID1). The costimulatory region may be derived from an intracellular portion of the transmembrane protein OX-40 (also known as tumor necrosis factor receptor superfamily member 4, TNFRSF4, RP5-902P8.3, ACT35, CD134, 0X40, TXGP1L). The costimulatory region may be derived from an intracellular portion of the transmembrane protein BTLA (also known as B- and T-Lymphocyte-Associated Protein, BTLA1 and CD272). The costimulatory region may be derived from an intracellular portion of the transmembrane protein CD27 (also known as S152, T14, Tumor Necrosis Factor Receptor Superfamily Member 7, TNFRSF7, and Tp55). The costimulatory region may be derived from an intracellular portion of the transmembrane protein CD30 (also known as tumor necrosis factor receptor superfamily member 8, TNFRSF8, D1S166E, and Ki-1). The costimulatory region may be derived from an intracellular portion of the transmembrane protein GITR (also known as tumor necrosis factor receptor superfamily member 18, TNFRSF18, RP5-902P8.2, AITR, CD357, ENERGEN, and GITR-D). The costimulatory region may be derived from an intracellular portion of the transmembrane protein HVEM (also known as tumor necrosis factor receptor superfamily member 14, TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, LIGHTR, and TR2). The costimulatory region may be derived from 2B4 (also known as CD244, NAIL, NKR2B4, Nmrk, SLAMF4, CD244 molecule, efc.). The costimulatory region may be derived from DNAM-1 (also known as CD226, DNAM1, PTA1, TLiSAl, CD226 molecule, efc.). The costimulatory region may be derived from CD40 (also known as Bp50, CDW40, TNFRSF5, p50, CD40 (protein), CD40 molecule, etc ). The costimulatory region may be derived from LFA-1 (also known as lymphocyte function-associated antigen 1, integrin alpha L, ITGAL, CD11A, LFA1A, integrin subunit alpha L, etc.). The costimulatory region may be derived from CD2 (also known as Lymphocyte-Function Antigen-2, LFA-2, SRBC, Til, CD2 molecule, etc.). The costimulatory region may be derived from CD7 (also known as GP40, LEU-9, TP41, Tp40, CD7 molecule, etc ). The costimulatory region may be derived from LIGHT (also known as TNFSF14, CD258, HVEML, LIGHT, LTg, TR2, TNLG1D, tumor necrosis factor superfamily member 14, etc.). The costimulatory region may be derived from NKG2C (also known as KLRC2, CD 159c, NKG2-C, killer cell lectin like receptor C2, etc.).

[0123] Specific exemplary co-stimulatory domains are represented by the amino acid sequences below:Table: Co-stimulatory domains302139246.1 - 36 -G. Detection Peptides

[0124] The CARs described herein may further comprise a detection peptide or molecule. Suitable detection peptides include hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:61); FLAG (e.g., DYKDDDDK; SEQ ID NO:62); c-myc (e.g., EQKLISEEDL; SEQ ID NO:63), and the like. Other suitable detection peptides are known in the art.H. Peptide Linkers

[0125] The polypeptides of the disclosure may include peptide linkers (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domain / regions described herein. As an example, a linker may be between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen302139246.1 - 37 -binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, flanking the costimulatory region or on the N- or C- region of the costimulatory region, and / or between the transmembrane domain and the endodomain. The peptide linker may have any of a variety of amino acid sequences. Domains and regions can be joined by a peptide linker that is generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins.

[0126] Peptide linkers with a degree of flexibility can be used. The peptide linkers may have virtually any amino acid sequence, bearing in mind that suitable peptide linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.

[0127] Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0128] Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0129] Example flexible linkers include glycine polymers (G)n, glycine- serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:64), (G4S)n and (GGGS)n (SEQ ID NO:65), where n is an integer of at least one. n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 66), GGSGG (SEQ ID NO: 67), GSGSG (SEQ ID NO: 68), GSGGG (SEQ ID NO:69), GGGSG (SEQ ID NO:70), GSSSG (SEQ ID NO:71), and the like. The linker may302139246.1 - 38 -comprise a repeat, such as a contiguous repeat of one or more of SEQ ID NOS:64-71, such as a linker comprising an amino acid sequence that corresponds to one of SEQ ID NOS:64-71, repeated at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or any range derivable therein.The linker may comprise (EAAAK)n (SEQ ID NO:72), wherein n is an integer of at least one. n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein).III. Obtaining Encoded Polypeptide Embodiments

[0130] In some aspects, there are nucleic acid molecule encoding polypeptides (e.g., antibodies, antibody fragments, CARs, TCRs, and bispecific molecules). These may be generated by methods known in the art, e.g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules.A. Expression

[0131] The nucleic acid molecules may be used to express large quantities of recombinant antibodies or to produce chimeric antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and other antibody derivatives. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization.1. Vectors

[0132] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigenbinding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody 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.

[0133] To express the antibodies, or antigen-binding fragments thereof, DNAs encoding partial or full-length light and heavy chains are inserted into expression vectors such that the302139246.1 - 39 -gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.2. Expression Systems

[0134] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.3. Methods of Gene Transfer

[0135] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each302139246.1 - 40 -incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No.5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by acoustofluidic gene delivery (see for example, WO2018148715 and Belling et al., PNAS 117 (20), 10976-10982, each of which are incorporated by reference for all purposes); 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; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by biophysical methods such as mechanical cell squeezing (Sharei et al., PNAS 2013 - 110 (6) 2082-2087; PNAS 2018 - 115 El 0907 and WO2017041050, both of which are incorporated by reference for all purposes), by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.4. Host Cells

[0136] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. Antibodies can be expressed in a variety of cell types. An expression construct encoding an antibody can be transfected into cells according to 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 employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NFAT-1 or NF-KB, both of which are 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 modulation of cytokine signaling, both in the T cells302139246.1 - 41 -themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0137] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.B. Isolation

[0138] The nucleic acid molecule encoding either or both of the entire heavy and light chains of an antibody or the variable regions thereof may be obtained from any source that produces antibodies. Methods of isolating mRNA encoding an antibody are well known in the art. See e.g., Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, e.g., Kabat et al., 1991, supra. Nucleic acid molecules encoding the full-length heavy and / or light chains may then be expressed in a cell into which they have been introduced and the antibody isolated.IV. Additional TherapiesA. Immunostimulators

[0139] In some embodiments, the method further comprises administration of an additional therapy or agent. In some embodiments, the additional therapy is an immunostimulator. The term “immunostimulator” as used herein refers to a compound that can stimulate an immune response in a subject, and may include an adjuvant. In some embodiments, an immunostimulator is an agent that does not constitute a specific antigen, but can boost the strength and longevity of an immune response to an antigen. Such immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli, Salmonella302139246.1 - 42 -minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.), liposomes and liposomal formulations such as AS01, synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments.

[0140] In some embodiments, the additional therapy comprises an agonist for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and / or combinations thereof. In some embodiments, additional therapies comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited immunostimulators comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381, U.S. Published Patent Application 2010 / 0075995, or WO 2010 / 018132; immunostimulatory DNA; or immunostimulatory RNA. In some embodiments, the additional therapies also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA, poly EC or poly I:poly C12U (available as Ampligen.RTM., both poly LC and poly LpolyC12U being known as TLR3 stimulants), and / or those disclosed in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280 A2. In some embodiments, an additional therapy may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some embodiments, additional therapies may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725.302139246.1 - 43 -

[0141] In some embodiments, additional therapies may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, additional therapies may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, additional therapies may be activated components of immune complexes. Additional therapies also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some embodiments, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some embodiments, immunostimulators are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. In some embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.B. Immunotherapies

[0142] In some embodiments, the additional therapy comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below.1. Inhibition of co-stimulatory molecules

[0143] In some embodiments, the immunotherapy comprises an inhibitor of a costimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.2. Dendritic cell therapy302139246.1 - 44 -

[0144] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0145] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0146] Dendritic cells can also be activated in vivo by making tumor cells express GM-CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0147] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (i.e. a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0148] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.3. CAR-T cell therapy

[0149] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0150] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells302139246.1 - 45 -is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.4. Cytokine therapy

[0151] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0152] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNI).

[0153] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.5. Adoptive T-cell therapy

[0154] Adoptive T cell therapy is a form of passive immunization by the transfusion of T-cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically, they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.302139246.1 - 46 -

[0155] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.6. Checkpoint Inhibitors and Combination Treatment

[0156] In some embodiments, the additional immunotherapy comprises immune checkpoint inhibitors. Certain embodiments are further described below.

[0157] PD -1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0158] 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 embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0159] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.302139246.1 - 47 -

[0160] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0161] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0162] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, 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 embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0163] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits302139246.1 - 48 -an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0164] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0165] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0166] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0167] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, 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 embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable302139246.1 - 49 -range therein) variable region amino acid sequence identity with the above-mentioned antibodies.C. Oncolytic virus

[0168] In some embodiments, the additional therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapyD. Polysaccharides

[0169] In some embodiments, the additional therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.E. Neoantigens

[0170] In some embodiments, the additional therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.F. Chemotherapies

[0171] In some embodiments, the additional therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate),302139246.1 - 50 -pyrimidine analogs (e.g., 5 -fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0172] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2 to about 20 mg / m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. In some embodiments, the amount of cisplatin delivered to the cell and / or subject in conjunction with the construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.

[0173] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter / TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and / or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.

[0174] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for an adult include about 60 mg / m2 to about 75 mg / m2 at about 21-day intervals or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.302139246.1 - 51 -

[0175] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0176] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0177] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0178] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows,302139246.1 - 52 -monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.G. Radiotherapy

[0179] In some embodiments, the additional therapy or prior therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0180] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any derivable range therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the does may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0181] In some embodiments, the amount of IR may be presented as a total dose of IR, which is then administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 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, 125, 130, 135, 140, or 150 (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein. In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,302139246.1 - 53 -19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.H. Surgery

[0182] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0183] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. 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. These treatments may be of varying dosages as well.V. Proteins and Nucleic Acid Compositions

[0184] As used herein, a “protein” “peptide” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. Wild-type versions of a protein or polypeptide may be employed, however, a modified protein or polypeptide may be employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a302139246.1 - 54 -protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. A modified / variant protein or polypeptide may have at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). A modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.

[0185] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solidphase peptide synthesis (SPPS) or other in vitro methods. Included in the disclosure are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0186] The size of a protein, polypeptide, or polynucleotides (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues or nucleic acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).

[0187] The protein, polypeptide, or nucleic acid may comprise an amino acid sequencing having, having at least, or having 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%,302139246.1 - 55 -85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) sequence identity to any one of SEQ ID NOS: 1-115.

[0188] The polypeptides, proteins, or polynucleotides of the disclosure may include or exclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions and / or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOS: 1-115. The peptide or polypeptide may be or may be based on a human sequence. The peptide or polypeptide may be not naturally occurring and / or is in a combination of peptides or polypeptides.

[0189] The substitution or variation may include or exclude a substitution or variation at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,302139246.1 - 56 -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, or 650 of any of SEQ ID NOS: 1-115 (or any derivable range therein) and may be or may exclude a substitution with any amino acid or may be or may exclude a substitution with a alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leusine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0190] The protein, polypeptide, or nucleic acid may comprise or may exclude 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, 118,302139246.1 - 57 -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 derivable range therein) of SEQ ID NOS: 1-115.

[0191] The protein, polypeptide, or nucleic acid may comprise or may exclude 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, 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 derivable range therein) of SEQ ID NOS: 1-115 and have or 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 derivable range therein) sequence identity to one of SEQ ID NOS: 1-115.302139246.1 - 58 -

[0192] The protein, polypeptide, or nucleic acid may comprise, comprise at least, comprise at most, or may exclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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 derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOS: 1-115.

[0193] The polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 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, 311,302139246.1 - 59 -312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any derivable range therein) contiguous amino acids of SEQ ID NOS: 1-115 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to one of SEQ ID NOS: 1-115.

[0194] The nucleic acid molecule or polypeptide may include a nucleic acid molecule or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,302139246.1 - 60 -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 of any of SEQ ID NOS: 1-115 and comprising at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 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, 240,302139246.1 - 61 -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, 886,302139246.1 - 62 -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 derivable range therein) contiguous amino acids or nucleotides of any of SEQ ID NOS: 1-115.

[0195] The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0196] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0197] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0198] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0199] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8,302139246.1 - 63 -9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type (or any range derivable therein). A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0200] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0201] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0202] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0203] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or302139246.1 - 64 -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 encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0204] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.

[0205] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. Areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0206] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a302139246.1 - 65 -similar biological activity. In making changes based upon the hydropathy index, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the invention, those that are within ±1 are included, and in other aspects of the invention, those within ±0.5 are included.

[0207] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (—0.5); histidine (—0.5); cysteine (—1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain aspects, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other aspects, those which are within ±1 are included, and in still other aspects, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0208] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0209] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such302139246.1 - 66 -residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein structure.

[0210] Amino acid substitutions may be made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. Conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).

[0211] Nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. Nucleic acids that encode the epitope to which certain of the antibodies provided herein are also provided. Nucleic acids encoding fusion proteins that include these peptides are also provided. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0212] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides can include regulatory sequences, isolated substantially away from their302139246.1 - 67 -naturally occurring genes or protein encoding sequences. Polynucleotides may be singlestranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0213] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0214] Included herein are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). The isolated polynucleotide may comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous302139246.1 - 68 -coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.VI. Administration of Therapeutic Compositions

[0215] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second cancer treatments are administered in a separate composition. In some embodiments, the first and second cancer treatments are in the same composition.

[0216] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0217] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0218] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous302139246.1 - 69 -infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0219] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0220] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0221] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment302139246.1 - 70 -(alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0222] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0223] The cancers amenable for treatment can include or exclude tumors of all types, locations, sizes, and characteristics. The cancer may comprise a solid tumor. The methods may include reducing tumor volume or treating cancers that are recurrent and / or metastatic. The cancer can comprise or exclude pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumors brain tumor, visual pathway and hypothalamic glioma, breast cancer, lymphoid cancer, bronchial adenomas / carcinoids, tracheal cancer, lung cancer, Burkitt lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal carcinoma of unknown primary, central nervous system lymphoma, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, 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 melanoma eye Cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, glioma of the brain stem, glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma,302139246.1 - 71 -childhood intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer , leukemia, acute lymphoblastic (also called acute lymphocytic leukemia) leukemia, acute myeloid (also called acute myelogenous leukemia) leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia) leukemia, chronic myelogenous (also called chronic myeloid leukemia) leukemia, hairy cell lip and oral cavity cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphomas, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Non-Hodgkin (an old classification of all lymphomas except Hodgkin's) lymphoma, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, childhood medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant, fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, childhood Salivary gland cancer Sarcoma, Ewing family of tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sezary syndrome sarcoma, skin cancer (nonmelanoma), skin cancer (melanoma), skin carcinoma, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, metastatic stomach cancer, supratentorial primitive neuroectodermal tumor, childhood T-cell lymphoma, testicular cancer, throat cancer, thymoma, childhood thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, childhood vulvar cancer, and wilms tumor (kidney cancer).302139246.1 - 72 -VII. Cellular TherapiesA. Cells

[0224] Certain embodiments relate to cells comprising polypeptides or nucleic acids of the disclosure. In some embodiments the cell is an immune cell or a T cell. “T cell” includes all types of immune cells expressing CD3 including T-helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T-regulatory cells (Treg) gamma-delta T cells, natural-killer (NK) cells, and neutrophils. The T cell may refer to a CD4+ or CD8+ T cell.

[0225] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0226] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell (e.g., peripheral blood stem cell) or progenitor cell obtained from an individual.

[0227] Cells of the present disclosure may comprise one or more therapeutic polypeptides or polynucleotides. In some embodiments, disclosed is a cell comprising one or more CAR polypeptides. In some embodiments, a cell comprises a CAR polypeptide comprising a tumor antigen binding domain. In some embodiments, a cell comprises a CAR polypeptide comprising an antigen binding domain described herein. Cells comprising a CAR polypeptide may, in certain embodiments, further comprise one or more additional therapeutic polypeptides and / or polynucleotides. Cells comprising a therapeutic polypeptide or polynucleotide of the present disclosure may further comprise one or more additional genetic modifications (e.g., genetic mutations, gene deletions, gene additions, etc.) which, in some embodiments, improve302139246.1 - 73 -the efficacy or safety of a therapeutic cell. Certain non-limiting examples of such genetic modifications are described in: Puig-Saus and Ribas. Gene editing: towards the third generation of adoptive T cell transfer therapies. Immuno-Oncology Technology. 2019 June 13; 1:19-26, incorporated by reference herein in its entirety.B. Cell Culture

[0228] In some embodiments, cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some embodiments, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured for 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, or 40 days (or any range derivable therein). The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some embodiments, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L-glutamine. Other embodiments may employ further basal media formulations, such as chosen from the ones above.

[0229] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above medium in order to support the growth of cells. A defined medium, however, also can be used302139246.1 - 74 -if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0230] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0231] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular embodiments, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a302139246.1 - 75 -cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.C. Cell Generation

[0232] Certain methods of the disclosure concern culturing the cells obtained from human tissue samples. In particular embodiments of the present disclosure, cells are plated onto a substrate that allows for adherence of cells thereto. This may be carried out, for example, by plating the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When the one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, in certain embodiments cells are introduced into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface, such embodiments encompass plating onto a substrate, which allows adherence of cells thereto.

[0233] Cells of the present disclosure may be identified and characterized by their expression of specific marker proteins, such as cell-surface markers. Detection and isolation of these cells can be achieved, for example, through flow cytometry, confocal fluorescence microscopy, ELISA, and / or magnetic beads. Reverse-transcription polymerase chain reaction (RT-PCR) may be used to quantify cell-specific genes and / or to monitor changes in gene expression in response to differentiation. In certain embodiments, the marker proteins used to identify and characterize the cells are selected from the list consisting of c-Kit, Nanog, Sox2, Heyl, SMA, Vimentin, Cyclin D2, Snail, E-cadherin, Nkx2.5, GATA4, CD105, CD90, CD29, CD73, Wtl, CD34, CD45, and a combination thereof.VIII. Sequences

[0234] The amino acid sequences throughout the application and those in the table below may include or exclude a Methionine at the beginning of the sequence, such as the position immediately before the first amino acid of the sequence. The nucleic acid sequences throughout the application and those in the table below may include or exclude a Methionine codon (atg) at the beginning of the sequence, such as the positions immediately before the first nucleic acid of the sequence. The nucleic acid sequences throughout the application and those in the table below may include or exclude a stop codon (tga) at the end of the sequence, such as the positions immediately after the last nucleic acid of the sequence.302139246.1 - 76 -302139246.1 - 77 -302139246.1 - 78 -302139246.1 - 79 -302139246.1 - 80 -302139246.1 - 81 -302139246.1 - 82 -302139246.1 - 83 -302139246.1 - 84 -IX. Detailed Embodiments

[0235] Embodiment one is a nucleic acid encoding: (i) a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR); and (ii) a TNFa polypeptide or functional fragment thereof.

[0236] Embodiment two is the nucleic acid of embodiment one, wherein the nucleic acid is an expression construct.

[0237] Embodiment three is the nucleic acid of any one of embodiments one or two, wherein the expression construct is a plasmid or a viral vector.

[0238] Embodiment four is the nucleic acid of embodiment three, wherein the viral vector is a vector derived from a retrovirus or a vector derived from a lentivirus.

[0239] Embodiment five is the nucleic acid of any one of embodiments one through four, wherein the nucleic acid encoding for the TNFa polypeptide comprises SEQ ID NO: 103 or a nucleic acid sequence that has at least 70% sequence identity to SEQ ID NO: 103.

[0240] Embodiment six is the nucleic acid of any one of embodiments one through four, wherein the TNFa polypeptide comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:73, a fragment of the amino acid sequence of SEQ ID NO:1 or 73, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 or 73, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO: 1 or 73.

[0241] Embodiment seven is the nucleic acid of embodiment five or embodiment six, wherein the nucleic acid encoding for the TNFa polypeptide encodes a methionine immediately upstream of the nucleic acid comprising SEQ ID NO: 103 or the nucleic acid sequence that has at least 70% sequence identity to SEQ ID NO: 103, or wherein the TNFa polypeptide comprises a methionine immediately upstream of the amino acid sequence of SEQ ID NO:1 or 73, fragment thereof, or amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or 73, or fragment thereof.

[0242] Embodiment eight is the nucleic acid of any one of embodiments one through seven, wherein the nucleic acid further encodes for a suicide gene product.

[0243] Embodiment nine is the nucleic acid of embodiment eight, wherein the suicide gene product is herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase,302139246.1 - 85 -P-lactamase, nitroreductase (NTR), carboxypeptidase A, split Cas9, truncated EGFR, or inducible caspase 9.

[0244] Embodiment ten is the nucleic acid of any one of embodiments one through nine, wherein the nucleic acid comprises one or more promoter(s) that direct the expression of the suicide gene product, CAR or TCR, and / or TNFa polypeptide.

[0245] Embodiment eleven is the nucleic acid of embodiment ten, wherein the promoter is constitutive.

[0246] Embodiment twelve is the nucleic acid of embodiment ten, wherein the promoter is conditional.

[0247] Embodiment thirteen is the nucleic acid of any one of embodiments one through twelve, wherein the TNFa polypeptide is derived from a human TNFa polypeptide.

[0248] Embodiment fourteen is the nucleic acid of any one of embodiments one through thirteen, wherein the nucleic acid encodes for a signal sequence.

[0249] Embodiment fifteen is the nucleic acid of embodiment fourteen, wherein the nucleic acid encodes for a signal sequence that is at the amino-terminus of the TNFa polypeptide, CAR, and / or TCR of the polypeptide expressed from the nucleic acid.

[0250] Embodiment sixteen is the nucleic acid of any one of embodiments one through fifteen, wherein the TNFa polypeptide, CAR, and / or TCR are expressed from one nucleic acid.

[0251] Embodiment seventeen is the nucleic acid of embodiment sixteen, wherein the nucleic acid encodes for a self-cleaving peptide or internal ribosome entry sequence (IRES), wherein the self-cleaving peptide or IRES is between the TNFa polypeptide and the TCR or CAR.

[0252] Embodiment eighteen is the nucleic acid of embodiment seventeen, wherein the self-cleaving polypeptide comprises SEQ ID NO:2, 5, 89, or 92, a fragment of the amino acid sequence of one of SEQ ID NO:2, 5, 89, or 92, or an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO:2, 5, 89, or 92, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:2, 5, 89, or 92.

[0253] Embodiment nineteen is the nucleic acid of any one of embodiments one through eighteen, wherein the TCR comprises an engineered TCR.

[0254] Embodiment twenty is the nucleic acid of any one of embodiments one through nineteen, wherein the TCR comprises a single chain TCR.

[0255] Embodiment twenty-one is the nucleic acid of any one of embodiments one through twenty, wherein the CAR or TCR comprises an anti-NY-ESO-1 CAR or TCR.302139246.1 - 86 -

[0256] Embodiment twenty -two is the nucleic acid of any one of embodiments one through twenty, wherein the CAR or TCR comprises an anti-GD2 or an anti-HPV CAR or TCR.

[0257] Embodiment twenty-three is the nucleic acid of embodiment twenty-two, wherein the anti-HPV CAR or TCR comprises an anti-HPV- 16 CAR or TCR.

[0258] Embodiment twenty-four is the nucleic acid of embodiment twenty -three, wherein the anti-HPV CAR or TCR comprises an anti-HPV- 16 E7 CAR or TCR.

[0259] Embodiment twenty-five is the nucleic acid of any one of embodiments nineteen through twenty-four, wherein the TCR comprises a TCR-alpha and a TCR-beta polypeptide.

[0260] Embodiment twenty-six is the nucleic acid of embodiment twenty-five, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 107-109, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 107-109, respectively.

[0261] Embodiment twenty-seven is the nucleic acid of embodiment twenty-five or embodiment twenty-six, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 104-106, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 104-106, respectively.

[0262] Embodiment twenty-eight is the nucleic acid of any one of embodiments twenty-five through twenty-seven, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises the amino acid sequence of SEQ ID NO:3, a fragment of the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:3.

[0263] Embodiment twenty-nine is the nucleic acid of any one of embodiments twenty-five through twenty-eight, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises the amino acid sequence of SEQ ID NO:4, a fragment of the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:4.

[0264] Embodiment thirty is the nucleic acid of embodiment twenty-five, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises a CDR1, CDR2, and CDR3 having the amino302139246.1 - 87 -acid sequences of SEQ ID NOS: 110-112, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 110-112, respectively.

[0265] Embodiment thirty-one is the nucleic acid of embodiment twenty-five or embodiment thirty, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 113-115, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:113-115, respectively.

[0266] Embodiment thirty-two is the nucleic acid of any one of embodiments twenty-five, thirty, or thirty-one, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises the amino acid sequence of SEQ ID NO:91, a fragment of the amino acid sequence of SEQ ID NO:91, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:91, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:91.

[0267] Embodiment thirty-three is the nucleic acid of embodiment twenty-five or any one of embodiments thirty through thirty -two, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises the amino acid sequence of SEQ ID NO:93, a fragment of the amino acid sequence of SEQ ID NO:93, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:93, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ IDNO:93.

[0268] Embodiment thirty-four is the nucleic acid of embodiment twenty -two, wherein the nucleic acid encodes an anti-GD2 CAR comprising an anti-GD2 scFv.

[0269] Embodiment thirty-five is the nucleic acid of embodiment thirty-four, wherein the anti-GD2 scFv comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that have the amino acid sequence of SEQ ID NOS:97-102, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:97-102, respectively.

[0270] Embodiment thirty-six is the nucleic acid of embodiment thirty -four or embodiment thirty-five, wherein the anti-GD2 scFv comprises a variable heavy chain region (VH) and variable light chain region (VL) and wherein the VH comprises the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:94.

[0271] Embodiment thirty-seven is the nucleic acid of any one of embodiments thirty-four through thirty-six, wherein the anti-GD2 scFv comprises a variable heavy chain region (VH) and variable light chain region (VL) and wherein the VL comprises the amino acid sequence302139246.1 - 88 -of SEQ ID NO: 96 or an amino acid sequence that has at least 80% sequence identity to SEQ IDNO:96.

[0272] Embodiment thirty-eight is the nucleic acid of any one of embodiments thirty-four through thirty-six, wherein the VH is 3' proximal to the VL.

[0273] Embodiment thirty-nine is the nucleic acid of any one of embodiments thirty-four through thirty-six, wherein the VL is 3' proximal to the VH.

[0274] Embodiment forty is the nucleic acid of any one of embodiments thirty-four through thirty-eight, wherein the anti-GD2 scFv comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:86.

[0275] Embodiment forty-one is the nucleic acid of any one of embodiments one through forty, wherein the nucleic acid is DNA or is RNA.

[0276] Embodiment forty-two is the nucleic acid of any one of embodiments one through forty-one, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO:72, 74, or 75, a fragment of the nucleic acid sequence of SEQ ID NO:72, 74, or 75, a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:72, 74, or 75, or a nucleic acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:72, 74, or 75.

[0277] Embodiment forty -three is the nucleic acid of any one of embodiments twenty-eight through forty-two, wherein the nucleic acid immediately preceding the nucleic acid encoding for SEQ ID NOs:3, 4, 91, 93, 94, 96, and / or 86 encodes for a methionine.

[0278] Embodiment forty-four is the nucleic acid of any one of embodiments one through forty-three, wherein the nucleic acid further comprises a region that encodes a signal peptide.

[0279] Embodiment forty-five is a polypeptide expressed from the nucleic acid of any one of embodiments one through forty-four.

[0280] Embodiment forty-six is a cell or population of cells comprising the nucleic acid of any one of embodiments one through forty-four or the polypeptide of embodiment forty-five.

[0281] Embodiment forty-seven is the cell or cells of embodiment forty-six, wherein the cell or cells express a polypeptide encoded by the nucleic acid.

[0282] Embodiment forty-eight is a cell or population of cells comprising a heterologous nucleic acid encoding a TNFa polypeptide.

[0283] Embodiment forty-nine is the cell or cells of embodiment forty-eight, wherein the nucleic acid encoding the TNFa polypeptide is an expression construct.

[0284] Embodiment fifty is the cell or cells of embodiment forty-nine, wherein the expression construct is a plasmid or viral vector.302139246.1 - 89 -

[0285] Embodiment fifty-one is the cell or cells of embodiment fifty, wherein the viral vector is a vector derived from a retrovirus or a vector derived from a lentivirus.

[0286] Embodiment fifty-two is the cell or cells of any one of embodiments forty-six through fifty-one, wherein the cell comprises a nucleic acid encoding for a suicide gene product.

[0287] Embodiment fifty-three is the cell or cells of embodiment fifty-two, wherein the suicide gene product is HSV-TK, PNP, CD, carboxypeptidase G2, cytochrome P450, linamarase, P-lactamase, NTR, carboxypeptidase A, split Cas9, truncated EGFR, or inducible caspase 9.

[0288] Embodiment fifty-four is the cell or cells of any one of embodiments forty-eight through fifty -three, wherein the nucleic acid comprises one or more promoter(s) that direct the expression of the suicide gene product and / or TNFa polypeptide.

[0289] Embodiment fifty-five is the cell or cells of embodiment fifty-four, wherein the promoter is constitutive.

[0290] Embodiment fifty-six is the cell or cells of embodiment fifty-four, wherein the promoter is conditional.

[0291] Embodiment fifty-seven is the cell or cells of any one of embodiments forty-eight through fifty-six, wherein the TNFa polypeptide is derived from a human TNFa polypeptide.

[0292] Embodiment fifty-eight is the cell or cells of any one of embodiments forty-eight through fifty-seven, wherein the TNFa polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:73, a fragment of the amino acid sequence of SEQ ID NO: 1 or 73, an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or 73, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO: 1 or 73.

[0293] Embodiment fifty-nine is the cell or cells of any one of embodiments forty-eight through fifty-eight, wherein the nucleic acid encodes for a signal sequence at the amino-terminus of the TNFa polypeptide.

[0294] Embodiment sixty is the cell or cells of any one of embodiments forty-eight through fifty-nine, wherein the cell further comprises a nucleic acid encoding a CAR or TCR.

[0295] Embodiment sixty-one is the cell or cells of any one of embodiments forty-eight through sixty, wherein the cell expresses the CAR, TCR, and / or TNFa polypeptide encoded by the heterologous nucleic acid.

[0296] Embodiment sixty-two is the cell or cells of any one of embodiments fifty through sixty-one, wherein the plasmid or viral vector has integrated into the cell’s genome.302139246.1 - 90 -

[0297] Embodiment sixty-three is the cell or cells of any one of embodiments sixty-one or sixty-two, wherein the CAR or TCR is expressed from a nucleic acid comprising a constitutive promoter that directs the expression of the CAR or TCR.

[0298] Embodiment sixty-four is the cell or cells of any one of embodiments sixty-one or sixty-two, wherein the CAR or TCR is expressed from a nucleic acid comprising a conditional promoter that directs the expression of the CAR or TCR.

[0299] Embodiment sixty-five is the cell or cells of any one of embodiments sixty through sixty-four, wherein the CAR or TCR comprises an anti-NY-ESO-1 CAR or TCR.

[0300] Embodiment sixty-six is the cell or cells of any one of embodiments sixty through sixty-four, wherein the CAR or TCR comprises an anti-GD2 or an anti-HPV CAR or TCR.

[0301] Embodiment sixty-seven is the cell or cells of embodiment sixty-six, wherein the anti-HPV CAR or TCR comprises an anti-HPV-16 CAR or TCR.

[0302] Embodiment sixty-eight is the cell or cells of embodiment sixty-seven, wherein the anti-HPV CAR or TCR comprises an anti-HPV-16 E7 CAR or TCR.

[0303] Embodiment sixty-nine is the cell or cells of any one of embodiments forty-six through sixty-eight, wherein the TCR comprises a TCR-alpha and a TCR-beta polypeptide.

[0304] Embodiment seventy is the cell or cells of embodiment sixty-nine, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 107-109, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 107-109, respectively.

[0305] Embodiment seventy-one is the cell or cells of embodiment sixty-nine or embodiment seventy, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 104-106, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:104-106, respectively.

[0306] Embodiment seventy-two is the cell or cells of any one of embodiments sixty-nine through seventy-one, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises the amino acid sequence of SEQ ID NO:3, a fragment of SEQ ID NO:3, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:3.

[0307] Embodiment seventy-three is the cell or cells of any one of embodiments sixty-nine through seventy-two, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide302139246.1 - 91 -and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises the amino acid sequence of SEQ ID NO:4, a fragment of SEQ ID NO:4, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:4.

[0308] Embodiment seventy-four is the cell or cells of embodiment sixty-eight or embodiment sixty-nine, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 110-112, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 110-112, respectively.

[0309] Embodiment seventy-five is the cell or cells of embodiment sixty-eight, sixty-nine, or seventy-four, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 113-115, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:113-115, respectively.

[0310] Embodiment seventy-six is the cell or cells of any one of embodiments sixty-eight, sixty-nine, seventy-four, or seventy-five, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises the amino acid sequence of SEQ ID NO:91, a fragment of SEQ ID NO:91, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:91, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:91.

[0311] Embodiment seventy-seven is the cell or cells of any one of embodiments sixty-eight, sixty-nine, or seventy-four through seventy-six, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-beta polypeptide comprises the amino acid sequence of SEQ ID NO:93, a fragment of SEQ ID NO:93, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:93, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:93.

[0312] Embodiment seventy-eight is the cell or cells of embodiment sixty-six, wherein the nucleic acid encodes an anti-GD2 CAR comprising an anti-GD2 scFv.

[0313] Embodiment seventy-nine is the cell or cells of embodiment seventy-eight, wherein the anti-GD2 scFv comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that have the amino acid sequence of SEQ ID NOS:97-102, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:97-102, respectively.302139246.1 - 92 -

[0314] Embodiment eighty is the cell or cells of embodiment seventy-eight or embodiment seventy-nine, wherein the anti-GD2 scFv comprises a VH and VL and wherein the VH comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:94.

[0315] Embodiment eighty-one is the cell or cells of any one of embodiments seventy-eight through eighty, wherein the anti-GD2 scFv comprises a VH and VL and wherein the VL comprises the amino acid sequence of SEQ ID NO:96 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:96.

[0316] Embodiment eighty -two is the cell or cells of any one of embodiments seventy-eight through eighty-one, wherein the VH is 3' proximal to the VL.

[0317] Embodiment eighty-three is the cell or cells of any one of embodiments seventy-eight through eighty-one, wherein the VL is 3' proximal to the VH.

[0318] Embodiment eighty-four is the cell or cells of any one of embodiments seventy-eight through eighty-two, wherein the anti-GD2 scFv comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence having at least 80% sequence identity to SEQ IDNO:86.

[0319] Embodiment eighty-five is the cell or cells of any one of embodiments forty-six through eighty-four, wherein the cell(s) comprise a T cell, a natural killer (NK) cell, a natural killer T cell (NKT), an invariant natural killer T cell (iNKT), stem cell, lymphoid progenitor cell, PBMC, bone marrow cell, fetal liver cell, embryonic stem cell, cord blood cell, or an induced pluripotent stem cell (iPS cell).

[0320] Embodiment eighty-six is the cell or cells of embodiment eighty-five, wherein the cell is a T cell or an NK cell.

[0321] Embodiment eighty-seven is the cell or cells of embodiment eighty-six, wherein the T cell comprises a naive memory T cell.

[0322] Embodiment eighty-eight is the cell or cells of embodiment eighty-seven, wherein the naive memory T cell comprises a CD4+ or CD8+ T cell.

[0323] Embodiment eighty-nine is the cell or cells of any one of embodiments eighty-six through eighty-eight, wherein the T cell comprises a T cell from a population of CD14 depleted, CD25 depleted, and / or CD62L enriched PBMCs.

[0324] Embodiment ninety is the cell or cells of any one of embodiments forty-six through eighty-nine, wherein the cells are a population of cells comprising 103-108 cells (e.g., from 103to 108cells).302139246.1 - 93 -

[0325] Embodiment ninety-one is a composition comprising the nucleic acid of any one of embodiments one through forty-four, the polypeptide of embodiment forty-five, or the cell(s) of any one of embodiments forty-six through ninety, wherein the composition is a pharmaceutically acceptable formulation.

[0326] Embodiment ninety -two is a method of making a cell comprising introducing into a cell the nucleic acid of any one of embodiments one through forty-four.

[0327] Embodiment ninety -three is the method of embodiment ninety-two, wherein the cell is infected with a virus comprising the nucleic acid.

[0328] Embodiment ninety-four is the method of embodiment ninety-three, wherein the virus comprises lentivirus or a lentiviral-derived virus or vector.

[0329] Embodiment ninety-five is the method of any one of embodiments ninety-two through ninety-four, wherein the cell is a T cell, NK cell, NKT cell, iNKT cell, stem cell, lymphoid progenitor cell, PBMC, bone marrow cell, fetal liver cell, embryonic stem cell, cord blood cell, or iPS cell.

[0330] Embodiment ninety-six is the method of embodiment ninety-five, wherein the cell is a T cell or an NK cell.

[0331] Embodiment ninety-seven is the method of embodiment ninety-six, wherein the T cell comprises a naive memory T cell.

[0332] Embodiment ninety-eight is the method of embodiment ninety-seven, wherein the naive memory T cell comprises a CD4+ or CD8+ T cell.

[0333] Embodiment ninety-nine is the method of any one of embodiments ninety-six through ninety-eight, wherein the T cell comprises a T cell from a population of CD14 depleted, CD25 depleted, and / or CD62L enriched PBMCs.

[0334] Embodiment one hundred is the method of any one of embodiments ninety-six through ninety-eight, wherein the cell is not yet a T cell or NK cell, the method further comprising culturing the cell under conditions that promote the differentiation of the cell into a T cell or an NK cell.

[0335] Embodiment one hundred one is the method of any one of embodiments ninety-two through one hundred, further comprising culturing the cell under conditions to expand the cell before and / or after introducing the nucleic acid into the cell.

[0336] Embodiment one hundred two is the method of embodiment one hundred one, wherein the cell is cultured with serum -free medium.302139246.1 - 94 -

[0337] Embodiment one hundred three is a method of treating a patient with cancer comprising administering to the patient an effective amount of the composition of embodiment ninety-one.

[0338] Embodiment one hundred four is a method for increasing anti-tumor immunity and / or tumor cell cytotoxicity in a patient having cancer, the method comprising administering to the patient an effective amount of the composition of embodiment ninety-one.

[0339] Embodiment one hundred five is the method of any one of embodiments one hundred three through one hundred four, wherein the composition comprises the cells of any one of embodiments forty-six through ninety.

[0340] Embodiment one hundred six is the method of any one of embodiments one hundred three through one hundred five, wherein the cancer comprises a solid tumor.

[0341] Embodiment one hundred seven is the method of any one of embodiments one hundred three through one hundred six, wherein cancer comprises a mesenchymal solid tumor.

[0342] Embodiment one hundred eight is the method of any one of embodiments one hundred three through one hundred five, wherein the cancer comprises melanoma, carcinoma, osteosarcoma, or sarcoma.

[0343] Embodiment one hundred nine is the method of any one of embodiments one hundred three through one hundred eight, wherein the cancer comprises aNY-ESO-l+, GD2+, or HPV+ cancer.

[0344] Embodiment one hundred ten is the method of any one of embodiments one hundred three through one hundred nine, wherein the subject has been determined to have a NY-ESO-1+, GD2+, or HPV+ cancer.

[0345] Embodiment one hundred eleven is the method of any one of embodiments one hundred three through one hundred ten, further comprising administering an additional therapy to the patient.

[0346] Embodiment one hundred twelve is the method of embodiment one hundred eleven, wherein the additional therapy comprises an immunotherapy, anti-angiogenic therapy, chemotherapy, surgery, radiotherapy, neoantigen therapy, or vaccination.

[0347] Embodiment one hundred thirteen is the method of embodiment one hundred eleven, wherein the additional therapy comprises immunotherapy and wherein the immunotherapy comprises immune checkpoint inhibitor therapy or bispecific T cell engagers.

[0348] Embodiment one hundred fourteen is the method of any one of embodiments one hundred three through one hundred thirteen, wherein the cells are autologous.302139246.1 - 95 -X. Examples

[0349] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1: TNF-alpha Armored TCR-T cells

[0350] There is a need to improve cellular immunotherapies for cancer. Genetically engineered T-cell immunotherapy is revolutionizing the landscape of cancer treatment, with sustained clinical responses seen in a wide variety of malignancies. Current approaches utilize ex vivo transduction of a patient’s T-cells to express either a cancer antigen-specific T-cell receptor (TCR) or a chimeric antigen receptor (CAR). These reinfused cells then create a focused anti -turn or response in a variety of cancer subtypes (1-10). However, while these treatments lead to dramatic clinical responses in many patients, a significant number of patients remain who do not respond at all to the initial infusion of transgenic T-cells (4, 10-12). The identification of biological factors which contribute to the functional capacity in cellular therapies, and how these factors differ between responders and non-responders to these therapies, represents an unmet need in cancer immunotherapy.A. Tumor necrosis factor-alpha secretion levels were associated with superior clinical responses to TCR-T cell therapy.

[0351] Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine with diverse roles in inflammation, immunity, and cell death. TNF-alpha has been shown to enhance T-cell effector function, promote T-cell survival, and improve antitumor immunity (13, 14).

[0352] TNF-alpha is primarily associated with promoting T-helper 1 (Thl) cytokine responses (IL-2, IFN-y), which play an important role in the defense against intracellular pathogens and cancer (19, 20). In contrast, Th2 immune responses are characterized by the production of cytokines such as IL-4, IL-5, and IL-13. While important for the defense against extracellular pathogens and allergic responses, Th2 cytokines are also immunosuppressive in the setting of T-cell directed immunity, and can inhibit the anti-cancer functionality of T-cell-302139246.1 - 96 -based immunotherapies (21-25). While TNF-alpha is primarily associated with promoting Thl immune responses (26-29), the effects of TNF-alpha on Th2 cytokine activity are complex and can be influenced by a range of factors, including the cell type and context of cytokine production. In some cases, TNF-alpha has been shown to suppress Th2 cytokine production. For example, TNF-alpha can inhibit the production of IL-4 and IL-5 by human T-cells, and can also inhibit the differentiation of Th2 cells in vitro (30, 31).

[0353] The inventors demonstrated that increased TNF-alpha secretion levels and cytokinetic polyfunctionality are associated with superior clinical response to transgenic TCR-T therapy directed against the tumor antigen NY-ESO-1 for the treatment of melanoma and sarcoma (FIGS. 1A-C). The inventors also showed that immunosuppressive Th2 cytokine activity was associated with inferior clinical response to therapy in this setting, and that TNF-alpha levels suppressed this Th2 activity (FIGS. 1D-E).B. Tumor necrosis factor-alpha-“armed” TCR vectors resulted in greater antigen-dependent cytokine secretion levels, and were associated with superior anti-tumor activity of transgenic TCR-T cells in vitro.

[0354] Given these unique results, the inventors designed a novel lentiviral vector which simultaneously encodes for both a transgenic TCR directed against the tumor antigen NY-ESO-1, as well as TNF-alpha (FIG.2). NY-ESO-1 is a cancer-testis antigen which is not expressed in somatic tissues, and TCRs targeting this antigen have shown dramatic objective response rates in a variety of solid tumors, including melanoma and sarcoma (5, 10, 12, 32). Transduction with a vector co-expressing a transgenic TCR and TNF-alpha (“TNF-alpha-armed” TCR vector) led to both expression of the tumor antigen-specific TCR, as well as increased expression of TNF-alpha and corresponding TNF-alpha secretion when the transgenic T-cell engaged with its target antigen (FIGS. 3A-D). These T-cells caused significantly greater tumor cell killing activity in vitro compared to control T-cells transduced with the TCR alone (FIG. 4A-B). In light of these findings, which showed improved in vitro antitumor activity of the TNF-alpha armed TCR-T cells, the inventors anticipate that the addition of transgenic TNF-alpha to TCR-T cell therapeutics will result in superior anti-tumor functionality and tumoral T-cell infiltration in vivo compared to T-cells transduced with a TCR alone.C. Evaluating the impact of transgenic TNF-alpha co-delivery with a transgenic TCR on T-cell anti-tumor functionality in vivo.302139246.1 - 97 -

[0355] Incorporation of TNF-alpha co-transduction in TCR adoptive cell therapies for solid tumors is an unexplored area. While supplementation with other cytokines (e.g., IL-12) has been studied in the setting of cellular therapies (33-35), TNF-alpha augmentation has never been explored in the setting of transgenic adoptive cell therapeutics. Furthermore, there has never been any attempt at designing a novel lentiviral vector that co-expresses both a tumor antigen-specific TCR and TNF-alpha to enhance antitumor efficacy. This disclosure is the first to generate a vector for co-expressing a tumor antigen-specific TCR and TNF-alpha to enhance anti-tumor efficacy in solid tumors.

[0356] Cell therapies targeting solid tumors remain underutilized, due to limited efficacy. The majority of clinical success in adoptive T-cell therapy has been achieved in hematological malignancies. However, solid tumors pose significant challenges due to the immunosuppressive tumor microenvironment, which limits T-cell function. This disclosure solves the problem of targeting solid tumors by enhancing T-cell function in the context of enhanced cytokinetic polyfunctionality.

[0357] The role of TNF-alpha in T-cell phenotype and function is complex and context-dependent. While TNF-alpha can enhance T-cell activation and effector function, the role of sustained exposure to enhanced levels of this cytokine in the tumor microenvironment remains un-characterized. This disclosure provides compositions and methods to understand the mechanisms underlying TNF-alpha signaling in transgenic T-cell therapies, via TNF-alpha-armed TCR vectors, and provides a newer generation of cellular immunotherapies. For example, the addition of transgenic TNF-alpha to TCR-T cell therapeutics via a TNF-alpha-armed TCR vector will result in superior anti-tumor functionality in vivo compared to T-cells transduced with a TCR alone.

[0358] The following examples can show the impact of TNF-alpha-armed TCR vectors on transgenic T-cell immunotherapy for solid tumors, both in terms of overall effectiveness, as well as its impact on T-cell phenotype and functionality. In some examples, the impact of transgenic TNF-alpha co-delivery with a transgenic TCR on T-cell anti-tumor functionality can be assessed in vivo. In some examples, the impact of increased TNF-alpha secretion on enhancing T-cell functional phenotype and the associated with underlying signaling mechanisms can be determined. The inventors anticipate that the addition of transgenic TNF-alpha to TCR-T cell therapeutics via a TNF-alpha-armed TCR vector will result in superior anti-tumor functionality in vivo compared to T-cells transduced with a TCR alone.302139246.1 - 98 -

[0359] The inventors demonstrated that T-cells transduced with a TNF-alpha-armed NY-ESO-1 TCR vector display superior TNF-alpha secretion levels and cytokinetic polyfunctionality, as well as superior tumor cell killing in vitro (FIGS. 3-4).

[0360] In some examples, melanoma tumor cell lines (e.g, M407, M247-A2, and A375) can be used, which are HLA-A:02:01 positive, strongly positive for the tumor antigen NY-ESO-1, and responsive to NY-ESO-1 directed TCR-T-cells in vitro and in vivo (FIG. 5B; see e.g., 36, 37). The cells can be utilized in a murine xenograft model of transgenic TCR-T therapy (FIG. 5A) to test the effectiveness of TNF-alpha-armed NY-ESO-1 TCR / / / vivo. This approach can enable assessment of the effects on tumor growth and survival, as well as the degree of T-cell infiltration and tumor response to the transgenic T-cells. In some examples, a previously established (36, 37) mouse xenograft model of NY-ESO-1 TCR-T cell therapy (FIG. 5A-B) can be used to test the relative effectiveness of TNF-alpha-armed TCR vectors against a control vector encoding for the NY-ESO-1 TCR alone.

[0361] In some examples, commercially available human donor T-cells can be activated and transduced in three groups: 1) mock lentivirus, 2) NY-ESO-1 TCR lentivirus, and 3) TNF-alpha-armed NY-ESO-1 TCR lentivirus. As the addition of another transgene to the vector could reduce inherent transduction efficiency, a transduction enhancer may be used for transduction (e.g., LentiBOOST®, a universal poloxamer-based, receptor-independent adjuvant which facilitates fusion of lentivirus particles with cell membrane, significantly increasing transduction efficiency by ~3-fold in human peripheral blood mononuclear cells (38). Following transduction, T-cells can be expanded ex vivo using previously established OKT3 / IL-2 culture conditions (see e.g., 4, and 10, both of which are incorporated in their entirety herein by reference). Expression of the heterologous TCR on the surface of cells and TNF-alpha can be characterized by a previously described major histocompatibility complex (MHC) dextramer flow cytometry approach (see e.g., 4, 39, both of which are incorporated in their entirety herein by reference) and qRT-PCR, respectively. In instances where NY-ESO-1 TCR surface expression differs between cell preps by >5%, mock-infected T-cells can be added to the cell composition to normalize the proportion of TCR+cells. Cells can then be frozen until ready for use.

[0362] In some examples, 8-12-week-old immunodeficient mice (e.g., NSG™ non-obese diabetic (NOD) / severe combined immunodeficiency (SCID) / / / 2rg mice from The Jackson Laboratory®) can be implanted with tumors by subcutaneous injection of 1 x 106A375 human melanoma cells into a shaved right flank. A375 cells are strongly positive for NY-ESO-1 and302139246.1 - 99 -HLA-A:02:01, enabling the tumor cells to present NY-ESO-1 in a way that is recognized by the transgenic TCR, which is HLA-A:02:01 -restricted.

[0363] In some examples, tumor size can be assessed seven days post-tumor implantation. Mice with tumor volumes between 15-30 mm3can be randomly assigned to the three different treatment groups and 3 x 106T-cells can be injected retro-orbitally. Tumor length and width can be measured every 2-3 days using electronic calipers and volume can be calculated as v = 1 / 6 x TI x length x width x (length + width) / 2. Mice can be euthanized if weight loss exceeds 25% of total initial body weight or if signs of inhumane suffering are manifested.

[0364] The primary endpoint for this study may be the comparison of tumor growth between the three groups. To assess statistical significance, the inventors can use ANOVA with two-tailed post-hoc tests adjusting for multiple comparisons (e.g., Tukey-Kramer) to compare the mean tumor volumes between the groups. The significance level can be set at 0.05. Effect sizes (e.g., Cohen's f or Cohen’s d) can be calculated to evaluate the magnitude of differences between groups. Additionally, the inventors can use survival analysis e.g., Kaplan-Meier curves) to describe differences in survival between groups, and regression-based models, with a primary covariate indicating treatment group, to compare tumor growth rates between the groups. Statistical analysis can be performed on SAS and GraphPad Prism. Assuming a two-sided alpha level of 0.05 and an equal allocation between groups, there is 80% power to minimally detect a standardized effect size (e.g., Cohen’s f) of 0.55 with 12 mice per group, using the proposed analytical methods. Using a Bonferroni-adjusted alpha of 0.017 as an adjustment for multiple comparisons, 12 mice per group yields 80% power to minimally detect a standardized effect size (e.g., Cohen’s d) of 1.4 using a two-sample t-test as a conservative approximation of post-hoc comparisons. To account for potential variability in the tumor growth rates and response to treatment, the inventors can increase the sample size to 16 mice per group, for a total of 48 mice. This sample size should provide sufficient power to detect differences between the groups and account for potential dropouts or exclusions due to illness or technical issues. The inventors can also monitor the tumor growth rates and adjust the sample size if necessary, using an adaptive design approach to ensure adequate power throughout the study.

[0365] The inventors anticipate that the addition of transgenic TNF-alpha to TCR-T cell therapeutics via a TNF-alpha-armed TCR vector will result in superior tumoral T-cell infiltration and tumor cell death signaling in vivo compared to T-cells transduced with a TCR alone.302139246.1 - 100 -

[0366] To interrogate the direct infiltration of the tumor microenvironment and direct antitumor effects of the secreted effector cytokines, spatial transcriptomic profiling can be performed. Spatial transcriptomics has the advantage of preserving the spatial orientation that is critically important for studying cellular immunotherapies and their interactions within the tumor microenvironment (which can otherwise be lost in the setting of bulk sequencing approaches). The inventors have previous experience and expertise in utilizing the GeoMx digital spatial profiling platform (NanoString) to address these questions in the setting of TCR-T cell therapy for solid tumors (FIG. 6A-B) (40). This platform enables spatially resolved profiling of both RNA and protein targets within formalin-fixed paraffin-embedded (FFPE) tissue sections, allowing the inventors to examine the molecular characteristics of different areas within the tumor microenvironment. The inventors can utilize spatial transcriptomics to quantify different T-cell infiltrates within the tumor microenvironment, as well as gene expression patterns of the T-cells and the adjacent tumor cells. The inventors have shown that increased TNF-alpha exposure is associated with increased p-STATl and p-JNK signaling and with melanoma cell death / apoptosis in vitro (FIG. 7A-B).

[0367] In some examples, spatial transcriptomics (e.g., the GeoMx platform from NanoString) can be used to analyze gene expression patterns in tumor samples collected from tumor bearing mice treated with engineered T-cells expressing a transgenic TCR and TNF-alpha. The spatial profiling analysis can enable characterization of T-cell infiltration patterns, as well as gene expression patterns from both tumor cells and T-cells, as well as the degree to which T-cell infiltration correlates with any differences in tumor cell gene expression.

[0368] In some examples, experiments can be perform as previously reported (see e.g., 40, incorporated in its entirety herein by reference ). In some examples, formalin-fixed paraffin-embedded (FFPE) tumor sections can be cut at 5 pm thickness, mounted on glass slides, and deparaffinized using xylene and rehydrated using a graded ethanol series. Tumor sections can be stained with hematoxylin and eosin (H&E) to identify regions of interest for analysis. In some examples, the spatial transcriptomics assay (e.g., GeoMx digital) can be performed according to the manufacturer's instructions, e.g., fluorescent markers can be applied to slidemounted FFPE tissue sections (e.g., NY-ESO-1 (tumor marker) CD45 (lymphocyte marker), and Syto 83 nuclear stain), images can be acquired at 20X magnification and used to identify regions of interest (ROI) that include regions with varying degrees of CD45 expression. In some examples, ROIs can then be processed by microscope automation for UV-light cleavage of indexed oligos, and photocleaved oligos can be collected and hybridized for analysis on the NanoString nCounter Analysis system. In some examples, the assay can include positive and302139246.1 - 101 -negative controls for internal housekeeping genes to ensure assay quality. In some examples, following the spatial transcriptomics assay (e.g., GeoMx DSP), raw data can be generated in the form of image files and digital gene expression (DGE) tables. In some examples, the data can be normalized by the area and positive control signal (using External RNA Controls Consortium reference material) and gene targets can be annotated by the MSigDB Gene Sets. In some examples, the most recurrently annotated gene sets can be chosen as a single annotation for each target and differential gene expression analysis can be performed using the limma package in R. In some examples, gene expression data can be compared between groups of interest (e.g.., tumors from mice treated with: 1) mock transduced T-cells, 2) TCR-only-transduced T-cells, and 3) TNF-alpha-armed TCR transduced T-cells). In some examples, the inventors can adjust the significance level using the Bonferroni correction method to account for multiple comparisons. In some examples, the inventors will consider genes with a Bonferroni-corrected q < 0.05 and fold change > 2 to identify significant differences. In some examples, additional analyses of selected differentially regulated genes can be performed using multiplex immunofluorescence on a Vectra Polaris platform to validate the findings from the spatial transcriptomics platform e.g., GeoMx).

[0369] In some examples, T-cell marker gene expression levels (e.g., CD3, CD4, CD8, as well as cytokine and phenotypic markers) can be quantified within specific regions of interest within the tumor microenvironment within CD45-positive cells to examine the differences in T-cell infiltration patterns between treatment groups. Assuming a two-sided significance level of 0.017, after using Bonferroni to adjust for multiple comparisons between the three groups (mock transduced T-cells, NY-ESO-1 TCR-T-cells, and TNF-alpha-armed NY-ESO-1 TCR-T-cells), and a two-sample t-test, 16 tumors per treatment group yields 80% power to minimally detect a mean difference in T-cell infiltration levels of 2-fold between treatment groups, with a standard deviation of 1-fold.

[0370] In some examples, the inventors can measure differential protein and mRNA expression levels of tumor cells (i.e., within NY-ESO-1 -positive cells) within the tumor microenvironment (e.g., within both T-cell rich and T-cell poor areas) to examine the differences in gene expression patterns between treatment groups. Assuming a two-sided significance level of 0.017, after using Bonferroni to adjust for multiple comparisons between the three groups (mock transduced T-cells, NY-ESO-1 TCR-T-cells, and TNF-alpha-armed NY-ESO-1 TCR-T-cells), and a two-sample t-test, 16 tumors per treatment group yields 80% power to minimally detect a mean difference in gene expression levels of 2-fold between treatment groups, with a standard deviation of 1-fold.302139246.1 - 102 -

[0371] In some examples, the statistical considerations outlined above may be used to help ensure that the sample size for both T-cell infiltration and gene expression analysis is appropriate to detect significant differences between treatment groups. Additionally, the use of Bonferroni multiple comparisons correction (as aforementioned) can help minimize false positives and increase the robustness of the findings.

[0372] The inventors anticipate that the addition of transgenic TNF-alpha to TCR-T cell therapeutics will result in superior anti -turn or functionality and T-cell infiltration in vivo compared to T-cells transduced with a TCR alone. The inventors also anticipate slower rates of in vivo tumor growth and superior mouse survival in mice treated with TNF-alpha-armed NY-ESO-1 TCR transgenic T-cells compared to the control NY-ESO-1 TCR T-cells alone. Furthermore, the inventors anticipate that the mice treated with the TNF-alpha-armed NY-ESO-1 TCR transgenic T-cells will display increased T-cell infiltration and TNF-alpha secretion levels within the tumor microenvironment (TME), and their tumors obtained at necropsy will display increased STAT1 and JNK cell death signaling. These results are summarized in Table 1.D. Determining the impact of increased TNF-alpha secretion on enhancing T- cell functional phenotype and the associated underlying signaling mechanisms.

[0373] The inventors anticipate that the addition of transgenic TNF-alpha to TCR-T cell therapeutics will inhibit Th2 immunosuppressive cytokinetic activity.

[0374] The inventors have shown that immunosuppressive Th2 cytokine activity is associated with inferior clinical response to transgenic TCR-T-cell therapy and that exogenous TNF-alpha suppresses this Th2 activity in vitro (FIGs. 8A-B). The inventors have also demonstrated that TNF-alpha enhances Th9 activity in vitro (FIG. 8C). In some examples, the impact of sustained, increased TNF-alpha secretion levels from TNF-alpha-armed TCR302139246.1 - 103 -transgenic T-cells on Th2 phenotypic differentiation and cytokinetic polyfunctionality activities is assessed in vitro. These experiments can utilize complementary approaches, including, but not limited to, mass cytometry and single-cell barcode chip assay to interrogate cytokine secretion levels of T-cells at the single-cell level (e.g., IsoPlexis).

[0375] In order to simulate the effect of increased TNF-alpha secretion by transgenic TCR-T cell on T-cell phenotype and functionality within the tumor microenvironment, previously established repetitive stimulation assays can be utilized (FIG. 9), which approximate the sustained activity of the transgenic T-cells in response to their target tumor cells (52). In some examples, antigen-specific transgenic TCR-T cells (e.g., cells transduced with the TNF-alpha-armed NY-ESO-1 TCR or the control vector encoding the NY-ESO-1 TCR alone) can be cocultured with fresh target tumor cells (e.g., HLA-A:02:01 and NY-ESO-1 -positive melanoma lines, such as A375, M407, or M257-A2) at 1:1 effector-to-target ratio repeatedly every 48 hours. Every 48 hours, the TCR-T cells can be aspirated, counted, assessed for viability, and purified using anti-CD3 magnetic microbeads before being co-cultured with fresh tumor cells. TNF-alpha levels in the media can be assessed for each co-culture via ELISA. The inventors have shown that a TNF-alpha-armed NY-ESO-1 TCR vector yields greater exposure of TNF-alpha to the T-cells over time, as measured by area-under-the-curve metrics (FIGS. 10A-B).After five serial passages, T-cells can be collected and subjected to phenotypic analysis and single-cell cytokinetic polyfunctionality assays via mass cytometry (e.g., cytometry by time-of-flight (CyTOF®)) and single-cell barcode-chip assay (e.g., IsoPlexis), respectively. Classification of the CD4 subsets that can be interrogated, both via phenotypic markers and cytokinetic polyfunctionality, are outlined in Table 2.Table 2. Exemplary CD4 T-cell subset classification via mass cytometry and IsoPlexis assays.

[0376] In some examples, mass cytometry can be used to assess phenotypic changes in transgenic T-cells over time. This technique merges single-cell flow cytometry with mass spectrometry, enabling the monitoring of many more simultaneous factors than conventional302139246.1 - 104 -flow cytometry (53). Heavy metal-conjugated antibodies can be used to stain for various T cell phenotypic markers (e.g., CD45RA, CD45RO, CD62L, CCR7, CD 127, CD25, CD28, CXCR3, CCR4, CCR6, CCR10, CD4, and / or CD8), inhibitory surface markers (e.g., PD-1, CTLA-4, LAG-3, and / or TIM-3) and / or T-cell transcription factors (e.g., T-bet, and / or GATA3) in a single reaction. This can enable highly robust classification of different transgenic T-cell phenotypes over time in the setting of increased exposure to TNF-alpha arising from the TNF-alpha-armed TCR vector, including the different CD4 T-helper subtypes. This can also allow the inventors to explore phenotypic differentiation in both CD4 and CD8 linages over time, from the less terminally differentiated naive / stem cell memory / central memory T-cells, to the more terminally differentiated effector / exhausted T-cells. Data metaclustering of phenotypically similar cells can be performed using Cytofkit software (54, incorporated herein by reference in its entirety), and confirmatory manual gating can be performed using FlowJo software. The inventors have previously published using this technique and associated analyses (see e.g., 10), and have access to the Helios mass cytometry platform though the Jonsson Comprehensive Cancer Center Flow Cytometry core facility.

[0377] In some examples, a single-cell barcode chip assay can be used to determine cytokine secretion functionality of the transgenic T-cells, e.g., using the IsoPlexis single-cell barcode chip (SCBC), a microfluidic platform that enables quantification of up to 32 different cytokines secreted by 1000-2000 individual T-cells which have been stimulated by their target antigen in vitro (55, 56). Cells can be labeled with CD4 or CD8 antibodies following the repetitive stimulation assay with cognate antigen-expressing tumor cells as described above, and loaded onto the SCBC and assayed on an IsoSpark platform for single-cell cytokine secretion assays. CD4 cytokine profiles can be then categorized into Thl (IFN-y, IL-2), Th2 (IL-4, IL-5, IL-10, and IL-13), Th9 (IL-9), and Thl7 (IL-17A, IL-22) functionality profiles (as outlined in Table 2) and assigned a relative polyfunctional strength index (PSI) for these cytokine profiles, as previously described (55, 57, both of which are incorporated in their entirety herein by reference). The inventors have their own IsoPlexis platform for performing these experiments, and have previously published utilizing this technique (see e.g., 58).

[0378] Preliminary work by the inventors using mass cytometry and IsoPlexis systems indicated that 6-8 biological replicates for each condition are needed to detect a 30% difference in expression of phenotypic markers and cytokine PSI profiles, with 80% power and a two-sided alpha of 0.05, assuming a standard deviation of 15%. Statistical analysis can be302139246.1 - 105 -performed on SAS and GraphPad Prism. The inventors can utilize unpaired t-tests for our comparisons, and p-values <0.05 can be considered statistically significant.

[0379] The inventors anticipate that the impact of TNF-alpha on CD4 Th2 phenotype and functionality is mediated by tumor necrosis factor receptor 2 (TNFR2) signaling via modulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activity.

[0380] In some examples, a single-cell intracellular phospho-proteome barcode chip assay (e.g., IsoPlexis; FIG. 11), which is distinct from the single-cell cytokine secretome chip described above, can be used to simultaneously interrogate up to 15 phospho-proteins at the single-cell level (60, 61), at a greater resolution than traditional methods, such as western blotting. Using this approach, the inventors have demonstrated in a clinical cohort that NY-ESO-1 TCR-T-cells’ CD4 Th2 PSI levels are positively correlated with increased NF-kB pathway activity (FIG. 12), consistent with previously published results (30, 31). In some examples, the impact of increased TNF-alpha signaling from a TNF-alpha-armed NY-ESO-1 TCR vector on TNFR2 signaling utilizing the same repetitive stimulation assays described above can be assessed. In some examples, exogenous TNF-alpha treatment can be used with or without TNFR2 CRISPR / Cas9-mediated silencing as positive and negative controls.

[0381] In some examples, the same repetitive stimulation assays described above (TNF-alpha-armed NY-ESO-1 TCR transduced human T-cells compared to control NY-ESO-1 TCR transduced T-cells co-cultured with A375, M407, or M257-A2 human melanoma cells) can be utilized. After five serial passages, T-cells can be collected and subjected to IsoPlexis singlecell intracellular phospho-proteome barcode chip assay (60, 61). This assay can enable simultaneous single-cell quantification of signaling via multiple pathways known to be modulated by TNF-alpha (59, 62, 63), including NF-kB (e.g., p-IkBA, NF-kB p-p65), MAPK (e.g, p-MEKl / 2, p-ERKl / 2) and JAK / STAT (e.g, p-STATl, p-STAT3, p-STAT5), among others. Signaling pathway augmentations can be correlated with increased or decreased proportion of CD4 T-helper phenotypic / functionality classified cells (which will be quantified and characterized in parallel as outlined above). Signaling pathways’ association with the different T-helper subtypes is summarized in Table 3. In some examples, confirmatory western blot experiments can be performed on all differentially phosphorylated signaling molecules interrogated, and established pharmacologic inhibitors of the associated signaling molecules can also be used as controls (Table 3). In some examples, non-transduced T-cells and T-cells with TNFR2 CRISPR-mediated silencing can be treated with exogenous TNF-alpha to serve as positive and negative controls. The guide RNA sequences for TNFR2 have been previously described (see e.g., 64, incorporated in its entirety herein by reference), and the inventors have302139246.1 - 106 -shown effective silencing of TNFR2 expression in human peripheral mononuclear blood cells (FIG. 13)Table 3. CD4 T-cell subset classification via signaling pathway activity and associated pharmacologic inhibitors.

[0382] Data by the inventors from the IsoPlexis single-cell intracellular phospho-proteome barcode chip assay indicated that 5-6 biological replicates for each condition, with a two-sided alpha of 0.05 and assuming a standard deviation of 20%, gives 80% power to minimally detect a 40% difference in relative quantity of phosphorylated protein levels (as measured by functional strength index, i.e., the proportion of single cells from a given sample which are positive for a given phospho-protein multiplied by the overall signal intensity). Statistical analysis can be performed on SAS and GraphPad Prism, utilizing unpaired t-tests for comparisons, and p-values <0.05 will be considered statistically significant.

[0383] The inventors anticipate that the addition of transgenic TNF-alpha to TCR-T cell therapeutics, and the concomitant increases in TNF-alpha levels in the tumor microenvironment (as approximated via repetitive stimulation assays), will augment TNF-alpha / TNFR2 signaling through inhibition of the NF-kB pathway, leading to inhibition of Th2 immunosuppressive cytokinetic activity. The inventors also anticipate that repetitive stimulation assays with tumor cells expressing the target tumor antigen will lead to increased levels of TNF-alpha in the tumor microenvironment for those T-cells which have been transduced with a TNF-alpha-armed NY-ESO-1 TCR vector, which will lead to decreased proportion of CD4 Th2 T-cells as measured by phenotypic markers and cytokinetic polyfunctionality. In parallel, the inventors anticipate that decreased levels of CD4 Th2 T-cells will correlate with decreases in NF-kB pathway activity (e.g., p-IkBA and NF-kB p-P65), which will be mediated via TNF-alpha / TNFR2 signaling. The anticipated results are summarized in Table 4.302139246.1 - 107 -Table 4. Anticipated outcomes of CD4 Th2 phenotype, cytokinetic polyfunctionality, and NF-kB signaling for TNF-alpha-armed TCR and control TCR engineered T-cells.

[0384] Although the primary focus will be the relationship between increased TNF-alpha activity and decreased levels of CD4 Th2 T-cells as a mechanism for augmenting the T-cells’ antitumor activity, the inventors also consider the interrogation of multiple T-helper phenotypes simultaneously (as outlined in Table 2 and Table 3). The inventors anticipate that other novel associations between these phenotypes and TNF-alpha activity may be found.

[0385] Once the impact of increased TNF-alpha activity on CD4 T-helper phenotypes and associated functionality is characterized, the results can be cross-validated with the data from the GeoMx experiments on tumor xenografts treated with the different transgenic TCR vectors. In some examples, validation can be attempted with the results using multiplex immunofluorescence on the Vectra Polaris platform.

[0386] In some examples, conventional fluorescence flow cytometry, multiplexed sandwich ELISAs from co-culture media, and western blots of stimulated cell lysates, can be utilized.

[0387] Additional data related to this example can be found in Appendix I.Example 2: TNF-a-“armed” TCR-T and CAR-T cells enhance tumor control without toxicity

[0388] Engineered adoptive T cell therapies targeting cancer-specific antigens have transformed the treatment of blood cancers, but their activity against solid tumors has been limited. A major barrier is the inability of engineered T cells to sustain potent antitumor activity within the suppressive tumor environment without causing systemic toxicity. To address this challenge, the inventors engineered tumor-targeting T cell receptor (TCR) and chimeric antigen receptor (CAR)-T cells to have greatly enhanced secretion of the effector cytokine tumor necrosis factor alpha (TNF-a) in an exclusively antigen-dependent manner. Herein the inventors show that TNF- a -armed TCR / CAR-T cells achieve markedly improved tumor control across multiple solid tumor models (melanoma, cervical carcinoma, and osteosarcoma) without inducing systemic inflammation or end-organ damage. Mechanistically, enhanced302139246.1 - 108 -antitumor activity was associated with increased effector memory T cell infiltration into tumors, reduction of intratumoral regulatory T cells and myeloid-derived suppressor cells, and reduced VEGF-A / TGF-P immunosuppressive signaling within tumor tissue. Importantly, the increased TNF-a activity remained restricted to antigen-positive tumors, uncoupling therapeutic efficacy from toxicity. Together, these findings demonstrate a generalizable strategy for increasing the therapeutic index of engineered T cell therapies, and provide a framework for improving the durability and safety of cellular immunotherapy for solid tumors.

[0389] Genetically engineered T-cell immunotherapy is revolutionizing the landscape of cancer treatment, with sustained clinical responses seen in a wide variety of malignancies. Current approaches utilize ex vivo transduction of a patient’s T-cells to express either a cancer antigen-specific T-cell receptor (TCR) or a chimeric antigen receptor (CAR). These reinfused cells then create a focused anti-tumor response in a variety of cancer subtypes (1-10). However, while these treatments lead to dramatic clinical responses in many patients, durability of these responses is poor, and overall response rates are incomplete (4, 10-12). This is particularly true in solid tumors, which have lagged behind the success of cell therapies for hematologic malignancies. The identification of biological factors which contribute to the functional capacity of cellular therapies, and using such knowledge to improve new iterations of such therapeutics, represents an unmet need in cancer immunotherapy.

[0390] While TNF-a may have previously reported beneficial effects in T cells, TNF- a’s role in toxicity of cellular immunotherapies and immune checkpoint blockade therapies is well documented, making the concept of augmenting its secretion in these therapeutics potentially unsafe (14).

[0391] Herein the inventors describe the design and preclinical testing of novel lentiviral vectors which simultaneously encode for both a transgenic TCR or CAR directed against a wide variety of solid tumor antigens, as well as a supplemental copy of TNF-a. These TNF-a -“armed” TCR / CAR-T cells display increased expression of TNF-a mRNA and corresponding antigen-dependent TNF-a secretion. This approach takes advantage of the unique biology of TNF-a regulation in T-cells secondary to TCR stimulation, which occurs exclusively at the translational level, rather than transcriptional level, (25-27) thus enabling increased basal transcription of the gene which would theoretically not lead to translation and subsequent secretion in the absence of antigen stimulation. This phenomenon confers in the TNF-a-armed TCR / CAR-T cells superior antitumor functionality (increased effector cytokinetic activity against the tumor cells, increased intratumoral T-cell infiltration, and decreased intratumoral immunosuppressive cell types), while displaying no increases in systemic toxicity. Together,302139246.1 - 109 -these findings demonstrate a broadly applicable strategy to enhance the therapeutic index of engineered T cells in solid tumors by improving antitumor activity without compromising safety.A. RESULTS1. TNF-a-armed TCR / CAR-T cells display increased basal transcription of TNF-a while retaining only antigen-dependent increases in TNF-a secretion and increased antitumor activity in vitro.

[0392] PBMCs were transduced with 3rd generation lentiviruses encoding either a conventional TCR (against NY-ESO-1 or HPV16 E7) or CAR (against GD2) (Supplementary Data 1,2,3; Supplementary Figure 1,2,3) or variants which were designed to encode for the same TCR / CAR, along with a supplemental copy of TNF-a, which were termed TNF-a-armed TCR / CARs (Supplementary Data 4,5,6; Supplementary Figures 4,5,6). While all three TNF-a-armed variants displayed significant increases in basal (unstimulated) transcriptional activity of TNF-a (Figure 1A-C), they displayed significant increases in TNF-a secretion under antigen-dependent stimulation with corresponding tumor cell lines only (A375 melanoma, CaSki cervical carcinoma, and 143B osteosarcoma cells, respectively), while no significant differences in antigen-dependent interferon-gamma secretion activities were observed (Figure 1D-F). No significant differences in basal (unstimulated) secretion levels of TNF-a were observed in any of the products tested.

[0393] In order to assess the anti -tumor activity and TNF-a secretion activity longitudinally in vitro, the inventors utilized real-time fluorescent cell imaging of T-cell and tumor cell cocultures using the IncuCyte platform. Under chronic antigen stimulation conditions in vitro, TNF-a-armed TCR / CAR-T cells displayed both superior initial control of longitudinal cancer cell growth, as well as improved retention of tumor cell control over time, while the conventional TCR / CAR-T cells progressively lost the ability to control tumor cell expansion after repeated exposures (Figure 1G,I,K). This phenomenon coincided with significantly improved TNF-a secretion levels from the TNF-a-armed TCR / CAR-T cells which persisted robustly over time, while the conventional TCR / CAR-T cells eventually lost the ability to produced TNF-a under repeated exposures to tumor cells (Figure 1H,K,L).302139246.1 - 110 -2. Treatment with TNF-a-armed TCR / CAR-T cells results in improved tumor control and survival in vivo

[0394] In order to determine the translational impact of these products, the inventors tested how TNF-a-armed TCR / CAR-T cells performed in several preclinical in vivo models of adoptive T-cell therapy. First, the inventors engrafted xenograft tumors of IxlO6A375 melanoma cells (NY-ESO-1+, HLA-A*02:01+) into the flanks of immunodeficient NSG mice. Once tumors were established, HLA-A*02:01+ donor human T-cells engineered to express either the conventional 1G4 NY-ESO-1 TCR, or the TNF-a-armed variant of the same TCR were administered via retro-orbital injection. TNF-a-armed TCR-T cell treatment resulted in significantly slower tumor growth and improved survival compared to mice who were treated with conventional NY-ESO-1 TCR-T cells (Figure 2A,B). In parallel to the in vitro assays, the inventors recapitulated the approach using 4050 cervical carcinoma tumor xenografts (HPV16 E7+, HLA-A* 02:01+), which were also treated with TCR-T cells transduced with either the conventional or TNF-a-armed variants of the HPV16 E7 TCR. In keeping with the results observed with the NY-ESO-1 TCR system, the TNF-a-armed HPV16 E7 TCR-T cells resulted in significantly improved tumor control and survival (Figure 2C,D).

[0395] To establish whether or not this advantage of TNF-a-arming was also applicable in a mesenchymal solid tumor, which have generally been shown to have high inherent resistance to adoptive T-cell therapies, the inventors also replicated the same in vivo NSG mouse xenograft model utilizing 143B osteosarcoma cells (GD2+), which were then treated with either conventional GD2-CD28z CAR-T cells or the TNF-a-armed variant of the same GD2 CAR construct. While treatment with the conventional GD2 CAR-T cells resulted in no differences in tumor control or survival, treatment with the TNF-a-armed GD2 CAR-T cells lead to significantly slower tumor growth and improved survival (Figure 2E,F). Thus, TNF-a-arming of tumor antigen-specific TCR / CAR-T cells significantly enhanced their therapeutic efficacy against melanoma, carcinoma, and sarcoma solid tumor models.3. Treatment with TNF-a-armed TCR / CAR-T cells does not cause any increase in systemic or end-organ toxicity in vivo

[0396] Given the concern for potentially increased toxicity of treatment with a TNF-a-armed TCR / CAR-T cell product, the inventors performed extensive studies on overall mouse health, end-organ autoimmune infiltration, and serum cytokine dynamics of our in vivo adoptive cell therapy models described above. In all treatment / tumor conditions, the inventors302139246.1 - Ill -observed no significant differences in mouse temperature, weight, or body condition score over time when treated with either conventional TCR / CAR-T cells or TNF-a-armed TCR / CAR-T-cells (Supplementary Figure 7). Furthermore, no significant differences in serum levels of human TNF-a or IL-6 (which is associated with cytokine release syndrome) were observed over time between either treatment groups for a given TCR / CAR (Supplementary Figure 8).

[0397] Finally, the inventors performed necropsies of all mice at the time of sacrifice in order to evaluate differences in end-organ damage resulting from treatment with the TNF-a-armed TCR / CAR-T cells. Mouse colon, small intestine, lung, liver, and kidney tissues were sectioned and subjected to histopathological evaluation of lymphocytic infiltration and inflammatory damage to assess for autoimmune activity, as previously described (28, 29). Given the strong association between systemic elevations in TNF-a and inflammatory colitis / weight loss, the inventors evaluated for both evidence of inflammatory colitis and stool calprotectin levels longitudinally in all treatment cohorts, and found no significant differences in either inflammatory tissue damage / lymphocytic infiltration of mouse colons, or elevations in stool samples from mice treated with TNF-a-armed TCR / CAR-T cells when compared to the corresponding conventional TCR / CAR (Figure 3). In all three tumor systems tested, treatment with TNF-a-armed TCR / CAR-T cells was not associated with any significant increases in autoimmune activity of any of the other organs assessed (Supplementary Figure 9). Overall, these data demonstrate that TNF-a-armed TCR / CAR-T cells can improve antitumor efficacy and survival with no apparent increased safety risk in our preclinical modeling of this treatment.4. TNF-a-armed TCR / CAR-T cells are associated with increased tumor infiltrating lymphocytes, increased TNF-a signaling within tumors, decreased regulatory T-cells, and decreased myeloid- derived suppressor cells in vivo

[0398] In order to further interrogate the underlying changes in T-cell infiltration and tumor microenvironment remodeling occurring during treatment with TNF-a-armed TCR / CAR-T cells, the inventors performed quantitative immunohistochemistry, flow cytometry, multiplex cytokine ELISA, and tumor spatial transcriptomic profiling on the mice described above in our in vivo studies. Flow cytometry performed on peripheral T-cells at the time of sacrifice demonstrated that mice treated with TNF-a-armed TCR / CAR-T cells all displayed significantly reduced proportions of regulatory T-cells (Tregs) when compared to mice who302139246.1 - 112 -received treatment with the corresponding conventional TCR / CAR-T cells (Figure 4A,C,E). Furthermore, mice treated with TNF-a-armed NY-ESO-1 TCR-T cells or TNF-a-armed HPV16 E7 TCR-T cells displayed significant reductions in peripheral CD4 T-helper 2 (Th2) subsets (Figure 4A,C), which was not observed in the TNF-a-armed GD2 CAR-T cell treatment (Figure 4E). Consistent with the observation of reduced Treg proportions in all TNF-a-armed TCR / CAR-T cell treatments tested, the inventors observed significant reductions in serum IL- 10 levels in all mice treated with TNF-a-armed TCR / CAR-T cells, while the mice treated with TNF-a-armed NY-ESO-1 or HPV16 E7 TCR-T cells displayed significant reductions in serum levels of the Th2-associated cytokines IL-4 and IL-5, which was not seen in those mice treated with the TNF-a-armed GD2 CAR (Figure 4B,D,F). Finally, the inventors observed significant increases in several T-cell phenotypic populations in the TNF-a-armed TCR / CAR system when compared to the corresponding conventional TCR / CAR: the TNF-a-armed HPV16 E7 TCR system showed significant increases in CD4 naive and CD8 effector populations (Figure 4C), while the TNF-a-armed GD2 CAR system showed significant increases in the CD4 and CD8 central memory populations (Figure 4E).

[0399] Intratumorally, infiltration of human CD3+ T-cells was significantly increased in tumors from mice treated with TNF-a-armed TCR / CAR-T cells relative to the corresponding conventional TCR / CAR-T cells (Figure 5A). Furthermore, intratumoral proportions of Tregs were significantly reduced in all TNF-a-armed TCR / CAR-T cell treatments compared to the corresponding conventional TCR / CAR (Figure 5B,C). TNF-a-armed HPV16 E7 TCR also displayed a significant reduction of intratumoral CD4 Th2 cells as well, along with significantly reduced CD4 naive T-cells, while CD4 effector cells were increased (Figure 5B). In contrast to the TCR-T cell therapies tested, the TNF-a-armed GD2 CAR-T cells displayed a significant increase in intratumoral CD4 Thl7 cells, as well as significant increases in both CD4 and CD8 central memory T-cells (Figure 5C). Given the association between mesenchymal tumors and myeloid-derived suppressor cells (MDSCs) and their associated immunosuppressive activities which typically leads to such tumors being resistant to T-cellbased immunotherapy, (30) the inventors interrogated intratumoral granulocytic MDSC (G-MDSC) and monocytic MDSC (M-MDSC) proportions in the mice with osteosarcoma xenografts. The inventors observed significant reductions in M-MDSC proportions in tumors from mice treated with TNF-a-armed GD2 CAR-T cells (Figure 5D).

[0400] Spatial transcriptomic profiling revealed significant increases in TNF-a signaling via both p38 and NF-KB pathways within the tumor cells treated with TNF-a-armed TCR / CAR-T cells, suggestive of signaling via TNFR1 (Figure 6A). T-cell transcriptomic302139246.1 - 113 -profiling within the T-cells also revealed significantly increased TNF-a signaling via the NF-KB pathway only (Figure 6B). Furthermore, tumors treated with TNF-a-armed TCR / CAR-T cells demonstrated a significant reduction in a VEGFA / TGFB immunosuppressive gene expression pattern when compared the conventional TCR / CAR-T cell treated tumors (Figure 6C,D). Transcriptomic profiling of the T cells demonstrated an effector memory phenotype which predominated naive and stem cell memory / central memory phenotypes (Figure 6E,F), thus capable of cytotoxic properties whilst maintaining their proliferative potential, as exhibited through the expression of individual genes and proliferation-related pathways. Notably, while the cells were activated, they did not appear to be exhausted, as observed through their enrichment of effector T cell and PD1 low pathways (Figure 6G,H).B. DISCUSSION

[0401] Transgenic adoptive T-cell therapy approaches for solid tumors lack persistent antitumor activity, in stark contrast to their frequent and initially potent early objective clinical responses (4, 10, 31-33). This has been demonstrated both in investigational products, as well as in the recent FDA-approved cell afamitresgene autoleucel for synovial sarcoma (34) This widespread finding underscores the need to improve the persistence of the early anti-tumor activity of the transgenic T cells. The inventors sought to explore whether arming T cells with TNF-a secretion would be an effective and safe approach to augment these therapies for solid tumors.

[0402] Some previously studied cytokine-augmented adoptive T-cell products, such as the IL- 12 armored CAR, have been associated with profound clinical toxicity due to uncontrolled secretion of the cytokine payload (39). Therefore, it was unclear whether TNF-a armored CAR or TCR T cells would experience the same fate. The inventors hypothesized that encoding a supplemental copy of genomically identical TNF-a along with a tumor antigen-specific TCR or CAR would lead to increased basal transcription of the gene, while avoiding increased translation and secretion until proper antigen stimulation of the TCR or CAR. This baseline increased transcription would also allow for superior longitudinal antigen-dependent secretion of TNF-a, and avoid the rapid extinguishing of its secretion typically observed in normal chronic antigen stimulation, as the inventors observed in our studies.

[0403] The in vivo data revealed that TNF-a-armed TCR / CAR-T cells provided superior tumor control and survival in a wide variety of solid tumor types when compared to the conventional TCR / CAR. Importantly, this occurred in the absence of any apparent increase in302139246.1 - 114 -toxicity. The PBMC humanized NSG mouse system is a robust and sensitive system in order to study TNF-a-mediated toxicity. Human and mouse TNF-a share close homology, both human and mouse TNF-a are able to bind to and activate both species TNFR1 with striking similarity. Indeed, hTNF-a-transgenic NSG mice, possessing uncontrolled germline expression of human TNF-a, have been shown to rapidly develop cachexia / weight loss, inflammatory colitis, and have even been studied for the pathogenesis of rheumatoid arthritis (40-42). All of these pathologies are highly specific for TNF-a-mediated disease activity, and mirror these conditions as experienced by human subjects. All of the mice treated with TNF-a-armed TCR / CAR-T cells displayed no increased systemic TNF-a levels over time, nor did they develop any weight loss, cachexia, or any elevations in stool calprotectin or histologic evidence of inflammatory colitis. Furthermore, no increased incidence of end-organ autoimmune damage was seen in any of the constructs the inventors tested. These findings suggest that TNF-a-armed TCR / CAR-T cells may offer the benefit of increased antitumor activity without any increased risk of systemic or end-organ toxicity. Translation of these findings in clinical settings would also have the benefit of clinicians having access to a wide variety of anti-TNF-a biologies of varying therapeutic duration, which would provide an additional layer of safety in the event of any increased TNF-a activity leading to toxicity (14).

[0404] The impact of increase antigen-dependent TNF-a secretion activity within the T-cell populations observed both peripherally and within the tumor demonstrated significant reductions of a variety of immunosuppressive cell types which have been associated with impaired T-cell activity against solid tumors, including CD4 Tregs and Th2 cells and their corresponding cytokines. TNF-a acting through TNFR2, has been previously shown to be able to impair these T-cells’ differentiation and resultant immunosuppressive activity (20-24). Indeed, our previous studies on clinical TCR-T cell products for solid tumors demonstrated that lower levels of IL-10, IL-4, and IL-5 secretion indices were also associated with superior clinical response rates (13). Furthermore, the overall increase in intratumoral T-cells observed in treatment with TNF-a-armed TCR / CAR-T cells appeared to be more consistent with increases in T-cell proliferation signaling, as determined by spatial transcriptomic profiling, in keeping with previously published studies (14-19).

[0405] The impact of TNF-a on T-cell phenotypic differentiation which the inventors observed, reflected in increases in naive and central memory T-cells in mice treated with TNF-a-armed TCR / CAR-T cells, remains less clear. While presumably advantageous to the overall therapeutic impact of this product, given the well established connection between less terminally differentiated T-cells and therapeutic efficacy of T-cell-based immunotherapies302139246.1 - 115 -(43), and TNF-a signaling has been associated with the proliferation and activation of naive and memory subsets of T-cells (19), the underlying causes of variations in these phenomena in our different tumor and TCR / CAR systems the impact of TNF-a signaling via TNFR2 (or TNFR1 on T-cells) remains largely undefined. While outside the scope of the present study, our system provides a potential means by which to more deeply study these signaling and phenotypic differentiation programs in future studies, to better elucidate the impact of TNF-a signaling on T-cell biology within the TME.

[0406] The impact of increased TNF-a signaling on local MDSC proportions within the TME is similarly enigmatic. Previously published studies on this topic have generally shown that TNF-a signaling is a driver of MDSC recruitment to the TME (44, 45). However, Treg signaling is also a major factor which leads to MDSC recruitment to the tumor microenviroment (46-48). It is possible that, in this context, TNF-a activity leading to decreases in intratumoral Tregs predominate the overall holistic impact of MDSC trafficking to the TME. This also speaks to the fundamental difference between systemic elevations in TNF-a observed in a wide variety of cancers, and localized delivery under tumor antigen-specific conditions within the TME (14). Further studies will be needed to assess the different factors and TNF-a secretion activities which drive these disparate MDSC behaviors, as well as the complex interplay between MDSCs and Tregs.

[0407] In conclusion, the inventors have shown that TNF-a-armed TCR / CAR-T cell therapy is associated with significant improvements in antitumor control and survival in a wide variety of solid tumors, including melanoma, cervical carcinoma, and osteosarcoma. This therapeutic benefit occurred in the absence of any increased toxicity, and was associated with both increases in TNF-a-mediated tumor cell signaling, increased intratumoral T-cell infiltration, and modulation of the tumor microenvironment to favorably impact the local populations of immunosuppressive cell types such as Tregs, Th2 cells, and MDSCs. Together, these findings establish a broadly applicable approach to safely enhance the therapeutic index of engineered T cell therapies for solid tumors by a variety of complementary mechanisms.C. METHODS1. Cell Lines and Culture Conditions

[0408] All tumor cell lines were purchased from ATCC or donated by collaborators. All cell lines were cultured in RPMI 1640 (Thermo A1049101) supplemented with heat-inactivated 10%Fetal Bovine serum, lx Penecillin / Streptomycin (Thermo 15140122), lx GlutaMax302139246.1 - 116 -(Gibco 35050061). Tumor Lines are all HLA-A*02:01+unless otherwise noted, and are categorized as follows: melanoma cell lines A375 (HLA-A* 02:01+, NY-ESO-1+), M407 (HL A-A* 02:01+, NY-ESO-1+), and M257 (HLA-A*02:01-; NY-ESO-1+) were all donated. HPV16+ cervical carcinoma cell lines CaSki (HL A-A* 02:01+, HPV16-E7+) were purchased from ATCC-CRL-1550, while 4050 cells (HLA-A* 02:01+, HPV16-E7+) were a generous gift from Christian Hinricks. Osteosarcoma cell lines 143B (GD2+), G292 (GD2+), and MG63 (GD2+) were all purchased from ATCC. Peripheral blood mononuclear cells (PBMCs) were confirmed to be HLA-A*02:01 via flow. HLA-A*02:01-positive human PBMCs were donated by the Antoni Ribas laboratory (male) for use in NY-ESO1 and GD2 models, or purchased from Precision (cat#33000; female, Px#201844068) for use in HPV16 E7 models. Media for PBMC experiments used complete medias consisting of either AIM V (Gibco A3830801) or RPMI1640, supplemented with 10% heat inactivated human sera (Thermo: NC2028317) or fetal bovine sera, respectively, with additional supplements of lx Pen / Strep, and lx GlutaMax.2. Lentiviral Generation and Transduction of PBMCs

[0409] Lentiviruses used the pRRL backbone previously described (33). TNF alpha (TNF-a) armed PBMC lentivirus was generated by adding an optimized T2A sequence after the final coding region of the TCR beta chain or CAR, followed by TNF-a cDNA sequence. PBMCs were transduced and expanded ex vivo using our previously established OKT3 / IL-2 culture conditions (4, 10, 33). Briefly, frozen donor PBMCs were thawed and incubated in AIM V complete media supplemented with anti-CD3 OKT3 (50ng / mL)(Milteny 130-093-387) + 300IU / mL IL-2 (17ng / mL) (BioLegend 589104) for 48hours. Activated PBMCs were then transduced with Lentivirus at an MOI(15) with lx LentiBOOST for 24H at 1E6 cells / mL. After transduction, cells were harvested, washed, and expanded in complete AIM V media supplemented with IL-2 (17ng / mL) at le6 / mL for 4-6 days before harvesting for flow cytometry, co-culture, in vivo experiments, or cell freezing in CS10 (StemCell 07959). Viral copy number (VCN) was determined via digital droplet PCR (ddPCR) as previously described (33). Genomic calibrator SDC4 gene primers F: CAGGGTCTGGGAGCCAAGT (SEQ ID NO:77), R: Probe: GCACAGTGCTGGACATTGACA (SEQ ID NO:78). Lentivirus primers F: TAAGAGCAGCGGCCGGTCC (SEQ ID NO: 79), R: GTCTCTCAGCTGGTACACGGC (SEQ ID NO:80). FAM conjugates probes were used to detect viruses; Lentiviral probe: ACCAGCCTGATCGTGCACCCCTACA (SEQ ID NO:81). SDC4 calibrator probe was conjugated to HEX: CCCACCGAACCCAAGAAACTAGAGGAGAAT (SEQ ID NO:82).302139246.1 - 117 -3. Tumor Co-Culture Experiments

[0410] Co-culture experiments were performed as previously described (33). Briefly, PBMCs were thawed and recovered overnight in R10+IL2. The next day PBMCs were plated with tumor cell lines at a 1:1 cell ratio, and co-cultured in R10+IL2 for 20 hours. Cells were pelleted and supernatant collected for ELISA. For chronic antigen stimulation co-culture, tumor cells transduced with a nuclear RFP lentivirus were seeded in a 96-well plate 24 hours prior to PBMC addition at a T-cell Tumor cell ratio of 5:1. Plates were imaged every 2 hours for 72 hour stimulation cycles (4-5 cycles total) using an IncuCyte Zoom live-cell imaging platform (Essen Bioscience). Every 72 hours, the plate was centrifuged, media collected for downstream ELISA analysis, and 2E4 fresh tumor cells were added. The RFP+ object counts per well were recorded over time. Cancer cell growth was calculated as the count at any given time point, normalized by the count at t = Oh for a given 72 hour stimulation.4. In Vivo Tumor Models

[0411] NOD / SCID / gamma (NSG) mice were originally purchased from Jackson Laboratories and maintained in a local breeding colony. NY-ESO1 and GD2 antigen presenting models used 8-12 week old male mice, while HPV E7 presenting models use 8-12 week old female mice. Mice were seeded with tumors by subcutaneous injection into a shaved right flank of 1x106tumor cells expressing NY-ESO-1 (A375, melanoma), HPV-16 E7 (4050, cervical carcinoma), or GD2 (143B, osteosarcoma) in 100 pL RPMI1640. Following seven days of tumor growth, mice were randomly assigned to the three different treatment groups. 3 x 106T-cells (mock, conventional TCR / CAR, or TNF-a-armed TCR / CAR) were injected retro-orbitally. Mice had their temperature, weight, body condition score (53), and tumor length and width measured every 2-3 days. Tumor volume was calculated as v = 1 / 6 x TI x length x width x (length+width) / 2. 50 pL of blood was obtained retro-orbitally weekly and centriduged for 2000x g for 10 minutes in EDTA for plasma cytokine analysis via multiplexed sandwich ELISA as described below, and stool was collected weekly per cage for calprotectin analyses via ELISA. Mice were euthanized via cervical dislocation under isoflurane anesthesia when weight loss was > 25%, tumor ulceration occurred, or signs of inhumane suffering / distress are manifested. At the conclusions of the experiments / time of sacrifice, mice had their tumors excised and quartered for embedding in FFPE for downstream spatial profiling, or filtered through a mesh for isolation of tumor infiltrating lymphocytes, as discussed below. Furthermore, mice had their colon, small intestine, thyroid, liver, kidney,302139246.1 - 118 -pancreas, lungs, and heart embedded in FFPE and assessed for histologic evidence of lymphocytic infiltrates and inflammatory tissue damage, as described below. Following euthanasia, mice were also terminally bleed for flow cytometry of circulating PBMCs.5. Assessment of end-organ autoimmune damage

[0412] Following mouse sacrifice, selected tissues were evaluated for immune infiltrates on histology, as we have previously described (29). Briefly, harvested organs were fixed in 10% buffered formalin for at least 9 hours and then stored in 70% ethanol. Organs were embedded in paraffin, sectioned (4 pm), and stained with hematoxylin and eosin (H&E). Immune infiltration was quantified in tissues by blinded assessment on H&E sections (5 high powered fields / section in each animal) as previously described (29). Tissues evaluated included gut (colon, small intestine), lung, liver, heart, thyroid, kidney, salivary gland, and pancreas. Given the high concern for autoimmune colitis with TNF-a-based therapy, intestinal inflammation was evaluated by assessment of inflammatory cell infiltrate, epithelial cell changes, and mucosal architecture as previously reported in mouse models by Erben et al. (28). For other tissues (including lung, liver, kidney, salivary glands, pancreas, and thyroid), immune infiltrate was evaluated and scored as 0 (no immune infiltrate), 1 (1-2 focal areas of immune infiltrate and / or sparse interstitial inflammation), 2 [>2 focal areas of immune infiltration, and / or presence of both focal glomerulonephritis and perivascular immune infiltration (kidney), and / or diffuse immune infiltrate affecting >25-49% of the tissue area] or 3 (diffuse immune infiltrate affecting >50-75% of the tissue area).6. ELISA

[0413] Harvested media from cell co-cultures were incubated with IFN-y or TNF-a antibody as described by the manufacturer (R&D DIF50C, DTA00D). ELISA plates were washed and incubated with secondary HRP conjugated antibody, and visualized using a TMB substrate. Final amounts of cytokine per million cells were calculated using the standard curve provided and correcting values based on the cell amounts, TCR positivity as measured by flow cytometry, and dilution of supernatant used. For the calprotectin ELISAs, mouse stool samples from each treatment cage containing 3-5 mice each were collected and aggregated weekly and frozen at -80C. Following completion of the in vivo experiments, stool samples were manually homogenized and resuspended in sterile water, and were then subjected to calprotectin ELISA per the instructions of the manufacturer (Alpco, 30-6936).302139246.1 - 119 -7. Multiplex serum cytokine analysis

[0414] Mouse plasma from the in vivo assays described above was frozen until time for analysis, at which point it was thawed and loaded into Meteor chips (Bruker) according to the manufacturers’ instructions. Samples were analyzed in triplicate (8 pL total volume) with an antibody panel against human GM-CSF, IFN-y, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, and TNF-a on an IsoSpark platform (Bruker) and calibrated to internal standards. Outputs were averaged between triplicates and quantified in pg / mL.8. Flow Cytometry

[0415] Standard flow cytometry techniques were used to analyze T cell subtypes within the tumor microenvironment and peripheral blood. Tumors were harvested, mechanically disrupted by grinding over 40 pm nylon mesh into phosphate-buffered saline (PBS), which were then pelleted and stained with surface markers for 30 minutes in the dark at 4°C degrees. Terminal bleeds were performed into EDTA containing tubes, which were spun at 2000xg for 10 minutes to harvest sera. Remaining cell pellet was resuspended in ImL of ACK Lysis buffer and incubated at room temperature for 5 minutes. After incubation, 500 pL of FACS buffer was added to neutralize lysis buffer and spun for 400xg for 5 minutes. Supernatant was discarded and cell pellets were stained with master mix. Post staining incubation, samples were washed and fixed in 2% paraformaldehyde for analysis on the Attune NxT as part of the JCCC Flow Cytometry Core at UCLA. Antibodies for these studies were obtained from BD Horizon: CD127 (Clone HIL-7R-M21), CCR6 (Clone 11A9), BD Bioscience: CXCR3 (Clone IC61CXCR3), Biolegend: CD4 (Clone RPA-T4), TCR VB 13.1 (Clone H131), CD3 (Clone UCHT1), Invitrogen: CD25 (Clone BC96), CCR7 (Clone 3D 12), CD8 (Clone RPA-T8), CCR4 (Clone D8SEE), CD279 (Clone J105), CD45RA (Clone H100). CD4 T-helper cell phenotypes were defined as: T-helper 1: CD4+, CD25-, CD127-, CCR4-, CCR6-, CXCR3+; T-helper 2: CD4+, CD25-, CD127-, CCR4+, CCR6-. CXCR3- T-helper 9 :CD4+, CD25-, CD127-, CCR4-, CCR6+; T-helper 17: CD4+, CD25-, CD127-, CCR4+, CCR6+. CXCR3+. CD4 and CD8 differentiation state phenotypes were defined as: Naive: CCR7+, CD45RA+, Central Memory: CCR7+, CD45RA-, Effector Memory: CCR7-, CD45RA-, Effector: CCR7-, CD45RA+. MDSCs were evaluated using antibodies to Ly6C (clone HK1.4; Thermo), Ly6G (clone 1A8; Thermo), and CDllb (clone MI / 70; Thermo), and were defined as G-MDSC: CDllb+, Ly6G+, Ly6C-, M-MDSC: CDllb+, Ly6G-, Ly6G+.302139246.1 - 120 -9. Quantitative immunohistochemistry

[0416] Mouse tumor samples were embedded in formalin-fixed paraffin-embedded (FFPE) tissue blocks, sectioned at a thickness of 5 pm, and were stained for human CD3 at the UCLA Translational Pathology Core Laboratory. Slides were deparaffinized and rehydrated with a series of graded ethanols to deionized water. Antigen retrieval was performed in Tris-EDTA at pH 9, and slides were cooked at high pressure at 120 C for 5 minutes. Immunostaining was performed on Leica Bond III autostainers using Leica Bond ancillary reagents (Leica Biosystems). Human CD3 antibody (clone UCHT1) was utilized for staining. Antigenantibody binding was visualized via the REFINE polymer 3,30 diaminobenzidine (DAB) detection system (Lieca). Stained slides were treated with 0.5% cupric sulfate for 10 minutes, then counterstained with hematoxylin, and coverslipped for subsequent analysis. Digital image acquisition was performed using the Aperio ScanScope XT system and imported for quantitative analysis usi...

Claims

WHAT IS CLAIMED:

1. A method of treating a patient with cancer and / or for increasing anti-tumor immunity and / or tumor cell cytotoxicity in a patient having cancer comprising administering to the patient a population of cells comprising a heterologous nucleic acid encoding:(i) a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR); and (ii) a TNFa polypeptide or functional fragment thereof;wherein the cancer is a NY-ESO-1+, GD2+, or HPV+ cancer and wherein the CAR or TCR comprises an anti-NY-ESO-1, anti-GD2, or anti-HPV CAR or TCR.

2. A nucleic acid encoding:(i) a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR); and (ii) a TNFa polypeptide or functional fragment thereof.

3. The nucleic acid of claim 2, wherein the nucleic acid encoding for the TNFa polypeptide comprises SEQ ID NO: 103 or a nucleic acid sequence that has at least 70% sequence identity to SEQ ID NO: 103; and / or wherein the TNFa polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 73, a fragment of the amino acid sequence of SEQ ID NO: 1 or 73, an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or 73, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ IDNO:1 or 73.

4. The nucleic acid of claim 2 or 3, wherein the CAR or TCR comprises an anti-NY-ESO-1, anti-GD2, or anti-HPV CAR or TCR.

5. The nucleic acid of claim 4, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 107-109, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 107-109, respectively; and wherein the TCR-beta polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 104-106, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 104-106, respectively.

6. The nucleic acid of any one of claims 4 or 5, wherein the nucleic acid encodes for a NY-ESO TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises the amino acid sequence of SEQ ID NO:3, a fragment of the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:3; and wherein the TCR-beta polypeptide comprises the amino acid sequence302139246.1 - 128 -of SEQ ID NO:4, a fragment of the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:4.

7. The nucleic acid of claim 4, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 110-112, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 110-112, respectively; and wherein the TCR-beta polypeptide comprises a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOS: 113-115, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS: 113-115, respectively.

8. The nucleic acid of claim 4 or 7, wherein the nucleic acid encodes for a HPV16-E7 TCR-alpha polypeptide and TCR-beta polypeptide, wherein the TCR-alpha polypeptide comprises the amino acid sequence of SEQ ID NO:91, a fragment of the amino acid sequence of SEQ ID NO:91, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:91, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:91; and wherein the TCR-beta polypeptide comprises the amino acid sequence of SEQ ID NO:93, a fragment of the amino acid sequence of SEQ ID NO:93, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:93, or an amino acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO:93.

9. The nucleic acid of claim 4, wherein the anti-GD2 scFv comprises a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that have the amino acid sequence of SEQ ID NOS:97-102, respectively, or an amino acid sequence that has at least 75% sequence identity to SEQ ID NOS:97-102, respectively.

10. The nucleic acid of claim 4 or 9, wherein the anti-GD2 scFv comprises a variable heavy chain region (VH) and variable light chain region (VL) and wherein the VH comprises the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:94, and wherein the VL comprises the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence that has at least 80% sequence identity to SEQ IDNO:96.

11. The nucleic acid of any one of claims 4, 9, or 10, wherein the anti-GD2 scFv comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:86.

12. The nucleic acid of any one of claims 2-11, wherein the nucleic acid is DNA or is RNA.302139246.1 - 129 -13. The nucleic acid of any one of claims 2-12, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO:72, 74, or 75, a fragment of the nucleic acid sequence of SEQ ID NO:72, 74, or 75, a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:72, 74, or 75, or a nucleic acid sequence that has at least 70% sequence identity to a fragment of SEQ ID NO: 72, 74, or 75.

14. The nucleic acid of any one of claims 3-13, wherein the nucleic acid immediately preceding the nucleic acid encoding for SEQ ID NOs:3, 4, 91, 93, 94, 96, and / or 86 encodes for a methionine.

15. A polypeptide expressed from the nucleic acid of any one of claims 2-14.

16. A cell or population of cells comprising i) the nucleic acid of any one of claims 2-14 or the polypeptide of claim 15 or ii) a heterologous nucleic acid encoding a TNFa polypeptide and a heterologous nucleic acid encoding a CAR or TCR.

17. A composition comprising the nucleic acid of any one of claims 2-14, polypeptide of claim 15, or the cell(s) of claim 16, wherein the composition is a pharmaceutically acceptable formulation.

18. A method of making a cell comprising introducing into a cell the nucleic acid of any of claims 2-14.

19. A method of treating a patient with cancer and / or for increasing anti -tumor immunity and / or tumor cell cytotoxicity in a patient having cancer comprising administering to the patient an effective amount of the composition of claim 17.

20. The method of claim 18 or 19, wherein the cancer comprises melanoma, carcinoma, osteosarcoma, or sarcoma; and / or wherein the cancer comprises a NY-ESO-1+, GD2+, or HPV+ cancer.302139246.1 - 130 -

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