Receptors providing targeted costimulation for adoptive cell therapy

Abstract

The present invention relates to a chimeric costimulatory antigen receptor (CoStAR) and cells containing CoStAR that are useful in adoptive cell therapy (ACT). CoStAR can act as a modulator of cell activity that enhances the response to a defined antigen. The present invention also provides CoStAR protein, the nucleic acid encoding CoStAR, and its therapeutic use.

Description

[Technical Field]

[0001] Related Applications and Incorporation by Reference This application is a continuation-in-part of U.S. Application No. 17 / 807109, entitled "RECEPTORS PROVIDING TARGETED COSTIMULATION FOR ADOPTIVE CELL THERAPY," filed June 15, 2022, and both this application and No. 17 / 807109 claim priority to U.S. Provisional Application No. 63 / 301340, entitled "RECEPTORS PROVIDING TARGETED COSTIMULATION FOR ADOPTIVE CELL THERAPY," filed January 20, 2022, each of which is incorporated by reference in its entirety. Reference is made to U.S. Provisional Patent Applications Nos. 63 / 211,042 and 63 / 211,046, filed June 16, 2021. Reference is made to UK Patent Application No. 1900858.0, filed January 22, 2019, U.S. Patent Application No. 62 / 951,770, filed December 20, 2019, International Application No. PCT / GB2020 / 050120, filed January 20, 2020, and U.S. Provisional Applications Nos. 63 / 053,494 and 63 / 053,498, filed July 17, 2020.

[0002] The aforementioned applications and all documents cited therein or in the prosecution thereof ("Application Citation Documents"), and all documents cited or referenced in the Application Citation Documents, and all documents cited or referenced herein ("Documents Cited Herein"), and all documents cited or referenced in the Documents Cited Herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product mentioned in this specification or any document incorporated by reference herein, are hereby incorporated by reference and may be utilized in the practice of this invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] Sequence Listing Reference This application has been submitted with an electronic Sequence Listing. The Sequence Listing is provided in a file named INSTB.008WO.xml, created on January 18, 2023, and is 37,111,282 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. The present invention relates to variants of the TRAF domain. [Background technology]

[0004] Adoptive cell therapy (ACT), which uses autologous T cells to mediate cancer regression, has shown great promise in early clinical trials. Several common approaches have been taken, including the use of naturally occurring tumor-reactive or tumor-infiltrating lymphocytes (TILs) expanded ex vivo. In addition, T cells can be genetically engineered to retarget them to defined tumor antigens. This can be achieved through gene transfer of peptide (p)-major histocompatibility complex (MHC)-specific T cell receptors (TCRs) or synthetic fusions between tumor-specific single-chain antibody fragments (scFvs) and T cell signaling domains (e.g., CD3ζ), the latter termed chimeric antigen receptors (CARs). TIL and TCR transfer have proven particularly successful in targeting melanoma (Rosenberg et al. 2011, Morgan 2006), while CAR therapy shows great promise in treating certain B-cell malignancies (Grupp et al. 2013).

[0005] Costimulatory signals are useful for achieving potent CAR T cell expansion, function, survival, and antitumor activity. The success of CAR therapy in leukemia has been attributed, in part, to the incorporation of costimulatory domains (e.g., CD28 or CD137) into the CAR construct, which synergize with signals provided by CD3ζ to enhance antitumor activity. The basis for this observation relates to the classical signal 1 / signal 2 paradigm of T cell activation, where signal 1 provided by the TCR complex synergizes with signal 2 provided by costimulatory receptors, such as CD28, CD137, or CD134, enabling cells to undergo clonal expansion, IL2 production, and long-term survival without activation-induced cell death (AICD) associated with signal 1 alone. Furthermore, engagement of signal 2 enhances signals generated through signal 1, enabling cells to better respond to low-avidity interactions, such as those encountered during antitumor responses.

[0006] Targeted costimulation may have beneficial effects in non-CAR-based T cell therapies. For example, incorporating costimulatory domains into chimeric TCRs has been shown to enhance T cell responses to pMHC (Govers 2014). Furthermore, there is a movement to generate chimeric costimulatory receptors in which the signal 2-generating costimulatory domain is separated from the signal 1-generating CD3ζ (or other) portion of the receptor, thereby potentially reducing the opportunity for tonic signaling (Fisher et al. 2019). Tumor-infiltrating lymphocytes (TILs) utilize their endogenous TCRs to mediate tumor recognition, but it has not been possible to manipulate endogenous TCRs. TILs are therefore severely limited by the fact that tumor cells express very few costimulatory ligands. The ability to induce targeted costimulation of TILs, or indeed any other adoptive T cell therapy product, would be beneficial. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0007] The present invention provides a novel chimeric costimulatory antigen receptor (CoStAR) that binds to carcinoembryonic antigen (CEA), and cells containing or expressing CoStAR, which are useful in CAR and non-CAR-based T cell therapies, etc. The present invention uses cells expressing the novel chimeric costimulatory receptor, which provides a costimulatory signal to T cells upon binding to a defined disease-associated antigen, e.g., a tumor-associated antigen. There have been several reports in which split signal 1 and signal 2 have been used to drive antigen-specific responses in engineered T cells (Alvarez-Vallina & Hawkins 1996). However, none have utilized the full-length CD28 molecule. There are certain advantages to using a full-length receptor, e.g., CD28, as opposed to a truncated form. A full-length receptor can allow dimerization, allowing the receptor to function in its native form; indeed, chimeric antigen receptors cannot function optimally when expressed as monomers (Bridgeman et al. 2010).

[0008] In some embodiments, the CoStAR of the present invention induces signal 2 upon binding to a defined antigen, e.g., a disease-associated antigen or a tumor-associated antigen. The full-length CD28 molecule contains a motif crucial for its inherent function in binding to members of the B7 family of receptors. This is potentially dangerous in light of the CAR having CD28 and CD3ζ receptors in tandem; ligation of the CAR with B7 can trigger T cell activation; however, a receptor having only the signal 2 receptor has beneficial properties. In one aspect, the present invention provides a targeted chimeric costimulatory receptor (CoStAR) comprising an extracellular binding domain operably linked to a transmembrane domain, a first signaling domain, and a CD40 signaling domain or a signaling fragment thereof. The inventors have discovered that costimulatory receptors containing the CD40 signaling domain exhibit novel and improved activity profiles. The inventors have further discovered that variants of the TRAF2, TRAF2 / 3, and TRAF6-binding domains within CD40 modulate signal transduction.

[0009] In certain embodiments of the invention, the CD40 signaling domain comprises SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. In some embodiments, the CD40 signaling fragment comprises an SH3 motif (KPTNKAPH, SEQ ID NO: 35), a TRAF2 motif (PKQE, SEQ ID NO: 36, PVQE, SEQ ID NO: 37, SVQE, SEQ ID NO: 38), a TRAF6 motif (QEPQEINFP, SEQ ID NO: 39), a PKA motif (KKPTNKA, SEQ ID NO: 40, SRISVQE, SEQ ID NO: 41), or a combination thereof, or is the full-length CD40 intracellular domain. In some embodiments, one or more of the SH3, TRAF2, TRAF6, or PKA motifs of the CD40 signaling domain are mutated.

[0010] In some embodiments, the first signaling domain of CoStAR comprises a signaling domain or signaling fragment of a receptor, for example, a tumor necrosis factor receptor superfamily (TNFRSF) receptor, including, but not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), DAP10, NTKR, CD357 (GITR), or EphB6. In some embodiments, CoStAR comprises CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), DAP10, NTKR, CD357 (GITR), or EphB6. In embodiments, the first signaling domain comprises a CD40 signaling domain, and thus, CoStAR comprises elements of two CD40 signaling domains.

[0011] In some embodiments, CoStAR comprises a second signaling domain or fragment of a receptor, for example, a tumor necrosis factor receptor superfamily (TNFRSF) receptor, including, but not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. The first signaling domain or fragment, CD40 signaling domain or fragment, and second signaling domain or fragment can be in any order. Exemplary embodiments include, without limitation, CoStAR comprising CD28, CD137, and CD40 signaling domains, CD28, CD134, and CD40 signaling domains, CD28, CD2, and CD40 signaling domains, CD28, GITR, and CD40 signaling domains, CD28, CD29, and CD40 signaling domains, or CD28, CD150, and CD40 signaling domains.

[0012] In some embodiments, the extracellular antigen-binding domain of the CoStAR of the present invention (e.g., without limitation, the CEA-binding domain or the MSLN-binding domain) is operably linked to the transmembrane domain via a linker and / or spacer. In some embodiments, the linker comprises about 5 to about 20 amino acids. In some embodiments, the linker comprises AAAGSGGSG (SEQ ID NO: 18).

[0013] In some embodiments, the CoStAR of the present invention comprises a spacer comprising about 10 to about 250 amino acids that operably links the extracellular binding domain to the transmembrane domain. In some embodiments, the spacer comprises the extracellular sequence of CD8 or CD28 or a fragment thereof. In some embodiments, the CoStAR comprises a second extracellular binding domain. In some embodiments, the second binding domain comprises an extracellular ligand-binding domain derived from CD8 or CD28. In some embodiments, the spacer comprises one or more immunoglobulin domains or immunoglobulin constant regions. In some embodiments, the spacer comprises one or more immunoglobulin domains or immunoglobulin constant regions, for example, but not limited to, SEQ ID NO: 24. In some embodiments, the transmembrane domain of the CoStAR of the present invention comprises the transmembrane domain of a TNFRSF protein. In some embodiments, the transmembrane domain of the CoStAR of the present invention comprises the transmembrane domain of CD28 or CD8. In some embodiments, the transmembrane domain of the CoStAR of the present invention comprises the transmembrane sequence of CD28 or CD8.

[0014] The CoStARs of the present invention are useful for stimulating immune responses against selected targets expressing tumor-associated antigens (TAAs), such as, but not limited to, carcinoembryonic antigen (CEA), mesothelin (MSLN), or others. In some embodiments, the CoStARs comprise an antigen-binding fragment of an scFv of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, e.g., a fragment comprising one, two, three, four, five, or all six complementarity-determining regions (CDRs). In some embodiments, the CoStARs comprise an scFv of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In some embodiments, the CoStARs comprise an antigen-binding fragment of an scFv of SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, or SEQ ID NO:1191, e.g., a fragment comprising one, two, three, four, five, or all six CDRs. In some embodiments, the CoStAR comprises the scFv of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191.

[0015] According to the present invention, the extracellular binding domain may include, but is not limited to, an scFv, a peptide, an antigen-binding portion of an antibody, an antibody heavy chain variable domain, an antibody light chain variable domain, a single domain antibody, a CEA ligand, or an MSLN ligand. In some embodiments, the CoStAR of the present invention comprises a CD3ζ signaling domain, e.g., located at the C-terminus. In some embodiments, the CoStAR of the present invention comprises an N-terminal signal peptide, non-limiting examples of which include the signal peptides of oncostatin M (OSM), CD8α, CD2, interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and human IgGκ. In one aspect of the present invention, a nucleic acid encoding the CoStAR of the present invention is provided. The nucleic acid may be optimized, for example, codon-optimized for expression in a host cell. In a non-limiting embodiment, the nucleic acid is codon-optimized for expression in a human cell.

[0016] In some embodiments, a vector is provided that encodes and is capable of expressing a CoStAR of any embodiment of the present disclosure. In some embodiments, cells expressing CoStAR of any embodiment of the present disclosure are provided. In some embodiments, the cells express two or more CoStARs, for example, the cells express CoStAR that binds to CEA or MSLN and CoStAR that binds to FOLR1, or CoStAR that binds to CA125, such as, but not limited to, anti-CEA.CD28.CD40 and anti-CA125.41BB.CD40, or anti-MSLN.CD28.CD40 and anti-CA125.41BB.CD40. In some embodiments, the cells express CoStAR that binds to CEA and CoStAR that binds to PDL1, such as, but not limited to, anti-CEA.CD28.CD40 and PD1.CD28.CD40, or CoStAR that binds to MSLN and CoStAR that binds to PDL1, such as, but not limited to, anti-MSLN.CD28.CD40 and PD1.CD28.CD40.

[0017] In some embodiments, the cells engineered to express CoStAR of any embodiment of the present disclosure comprise alpha-beta T cells, gamma-delta T cells, regulatory T cells, TILs, NKT cells, or NK cells. In some embodiments, the cells engineered to express CoStAR of any embodiment of the present disclosure co-express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Some embodiments provide a method of producing a cell that expresses CoStAR, the method comprising transducing or transfecting a cell with a vector encoding and capable of expressing CoStAR of any embodiment of the present disclosure.

[0018] Some embodiments provide methods for preparing a cell population expressing CoStAR of any embodiment of the present disclosure by transducing or transfecting the cells, detecting expression of CoStAR, and enriching, expanding, and / or selecting for cells expressing CoStAR. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins.

[0019] Some embodiments provide methods of treating a disease in a subject by administering a cell population expressing CoStAR of any embodiment of the present disclosure. In some embodiments, the protein will comprise a variant CD40 and / or TRAF2, 2 / 3, or 6 domain disclosed herein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins.

[0020] In some embodiments, the sequence of consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, X4 is any amino acid, X5 is Q, X6 is C, E, or A, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid, and X 10 is any amino acid, and X 11 is any amino acid. In some embodiments, the protein is part of a TRAF domain. In some embodiments, the protein is part of a TRAF2 domain. In some embodiments, the protein is part of a TRAF2 / 3 domain. In some embodiments, the sequence is: X1 is S or P, X2 is V, H, or Y, X4 is I, Q, V, X7 is T or S, X8 is D, X9 is K, D, or G, and X 10 is T, S, G, or A, and X 11 is D, S, N, or D, or the sequence is at least 60% identical to a). In some embodiments, the protein comprises the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42299. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42377. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42378.

[0021] In some embodiments, the sequence of consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11wherein X1 is any amino acid, X2 is any amino acid, X3 is P, S, A, or T, X4 is any amino acid, X5 is Q or E, X6 is E, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid, and X 10 is any amino acid, and X 11 is any amino acid. In some embodiments, the protein is a portion of a TRAF domain. In some embodiments, the protein is a portion of a TRAF2 domain. In some embodiments, the protein is a portion of a TRAF2 / 3 domain. In some embodiments, the sequence is: X1 is P, A, M, T, S, R, V, H; X2 is F, A, L, I, T, V, G, C, A, F, P; X4 is K, V, I, H, A, Q, T, E, S; X7 is C, T, E, D, S, A; X8 is A, L, G, Y, I, Q, D, E; X9 is F, H, K, R, P, A, G, Y, E, N; and X 10 are R, G, E, K, A, S, D, C, C, P, V, and X 11 is S, C, D, P, W, T, A, G, E, or the sequence is at least 60% identical to a). In some embodiments, the sequence is a portion of the Traf2 / 3 sequence. In some embodiments, the protein comprises the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42300. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42379. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42380.

[0022] In some embodiments, a protein is provided that comprises the consensus C sequence or an amino acid sequence comprising X1X2X3X4X5X6, where X1 is P, X2 is any amino acid, X3 is E, X4 is any amino acid, X5 is any amino acid, and X6 is Ac / Ar. In some embodiments, the protein is part of a TRAF domain. In some embodiments, the protein is part of a TRAF6 domain. In some embodiments, the sequence comprises X2 is Q, P, E, V, T, or S, X4 is I, L, M, N, S, T, N, D, and X5 is N, D, R, S, G, or Y, or the sequence is at least 60% identical to a). In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6, SEQ ID NO: 42301. In some embodiments, the protein is of the sequence X1X2X3X4X5X6PDDL, SEQ ID NO: 42381. In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0023] In some embodiments, the amino acid sequence comprises: (a) one or more of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; (b) a sequence that is at least 70% identical to the sequence in a); or (c) a sequence that is at least 80% similar to a).

[0024] In some embodiments, the TRAF domain in the CD40 protein is a TRAF domain comprising one or more of: (a) SEQ ID NO: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; (b) a sequence that is at least 70% identical to the sequence in a); or (c) a sequence that is at least 80% similar to a). In some embodiments, the TRAF6 domain sequence comprises PQEINF, PEEMSW, PPENYE, or PQENSY. In some embodiments, the TRAF2 / 3 domain sequence comprises PVQET, TQEET, SKEET, AVEET, or PVQET. In some embodiments, the TRAF2 domain sequence includes SVQE, AVEE, or PEEE.

[0025] In some embodiments, the TRAF6 or TRAF2 / 3 domain comprises an amino acid sequence of one of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; (b) an amino acid sequence that is at least 70% identical to a); or (c) an amino acid sequence that is at least 80% similar to a). In some embodiments, the sequence is within the CD40 protein sequence for the protein, amino acid sequence, or TRAF domain of any one of the embodiments of the present disclosure. In some embodiments, the sequence is within the CD40 protein and is located in the corresponding TRAF2, TRAF2 / 3, and / or TRAF6 domain within the CD40 protein.

[0026] In some embodiments, consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, X4 is any amino acid, X5 is Q, X6 is C, E, or A, X7 is any amino acid, X8 is any amino acid, and X9 is any amino acid; 10 is any amino acid, and X 11 is any amino acid. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11, SEQ ID NO: 42299. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42377. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42378.

[0027] In some embodiments, consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, S, A, or T, X4 is any amino acid, X5 is Q or E, X6 is E, X7 is any amino acid, X8 is any amino acid, and X9 is any amino acid; 10 is any amino acid, and X 11 is any amino acid. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42300. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42379. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42380.

[0028] In some embodiments, a TRAF6 domain comprises the amino acid sequence of consensus C or X1X2X3X4X5X6, where X1 is P, X2 is any amino acid, X3 is E, X4 is any amino acid, X5 is any amino acid, and X6 is Ac / Ar. In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6, SEQ ID NO: 42301. In some embodiments, the protein is of the sequence X1X2X3X4X5X6PDDL, SEQ ID NO: 42381. In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0029] In some embodiments, the CD40 domain comprises one or more of the following amino acid sequences: (a) consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, X4 is any amino acid, X5 is Q, X6 is C, E, or A, X7 is any amino acid, X8 is any amino acid, and X9 is any amino acid; 10 is any amino acid, and X 11 is any amino acid; (b) a TRAF2 / TRAF3 domain having consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, S, A, or T, X4 is any amino acid, X5 is Q or E, X6 is E, X7 is any amino acid, X8 is any amino acid, and X9 is any amino acid; 10 is any amino acid, and X 11is any amino acid; (c) a TRAF6 domain comprising the amino acid sequence of consensus C or X1X2X3X4X5X6, where X1 is P, X2 is any amino acid, X3 is E, X4 is any amino acid, X5 is any amino acid, and X6 is Ac / Ar.

[0030] In some embodiments, the construct is a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain that binds to carcinoembryonic antigen (CEA) or mesothelin (MSLN), a transmembrane domain linked to the extracellular binding domain, a first signaling domain, and a second signaling domain, wherein the second signaling domain comprises a variant CD40 signaling domain or a signaling fragment thereof, wherein the variant CD40 comprises a variant TRAF2 / TRAF3 sequence, a variant TRAF6 sequence, or a variant TRAF2 sequence, and wherein the variant CD40 does not comprise SEQ ID NO: 42. In some embodiments, the first signaling domain comprises a signaling domain or a signaling fragment of CD28 or CD278 (ICOS). In some embodiments, the construct is as shown in Figure 88A. In some embodiments, the construct is as shown in Figure 88B. In some embodiments, the construct is as shown in Figure 88C. In some embodiments, the first signaling domain comprises a full-length costimulatory domain. In some embodiments, the extracellular binding domain is operably linked to the transmembrane domain via a linker and / or spacer. In some embodiments, the linker comprises about 5 to about 20 amino acids. In some embodiments, the linker comprises about 10 to about 250 amino acids. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from CD28, CD8, ICOS, DAP10, or NTRK. In some embodiments, the transmembrane domain comprises the transmembrane domain sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the extracellular binding domain comprises an scFv, a peptide, an antibody heavy chain variable domain, an antibody light chain variable domain, or a CEA ligand.

[0031] In some embodiments, the nucleic acid encodes CoStAR of any one of the embodiments of the present disclosure. In some embodiments, the vector comprises a nucleic acid of any one of the embodiments of the present disclosure. In some embodiments, the cell is a cell that expresses any one of the CoStAR embodiments of the present disclosure. In some embodiments, the cell comprises an alpha-beta T cell, a gamma-delta T cell, a regulatory T cell, a TIL, an NKT cell, or an NK cell. In some embodiments, the cell co-expresses a CAR or a TCR. In some embodiments, a method of making a cell of any one of the embodiments of the present disclosure, the method comprising transducing or transfecting the cell with a vector of any one of the embodiments of the present disclosure.

[0032] In some embodiments, a method for preparing a cell population expressing CoStAR of any one of the embodiments of the present disclosure, comprising detecting expression of CoStAR on the surface of cells transfected or transduced with a vector of alternative 62, and selecting cells identified as expressing CoStAR. In some embodiments, the cell population is enriched for cellular expression of CoStAR of any one of the embodiments of the present disclosure. In some embodiments, a method for treating a disease in a subject, the method comprising administering to the subject a cell according to any one of the embodiments of the present disclosure or a cell population according to any one of the embodiments of the present disclosure.

[0033] In some embodiments, the protein comprises the amino acid sequence of one of SEQ ID NOs: 510-519. In some embodiments, the protein comprises the sequence of SEQ ID NO: 510. In some embodiments, the protein comprises the sequence of SEQ ID NO: 511. In some embodiments, the protein comprises the sequence of SEQ ID NO: 512. In some embodiments, the protein comprises the sequence of SEQ ID NO: 513. In some embodiments, the protein comprises the sequence of SEQ ID NO: 514. In some embodiments, the protein comprises the sequence of SEQ ID NO: 515. In some embodiments, the protein comprises the sequence of SEQ ID NO: 516. In some embodiments, the protein comprises the sequence of SEQ ID NO: 517. In some embodiments, the protein comprises the sequence of SEQ ID NO: 518. In some embodiments, the protein comprises the sequence of SEQ ID NO: 519. In some embodiments, the protein comprises any of the constructs (CTP188-CTP202) shown in FIG. 92.

[0034] Accordingly, it is the purpose of this disclosure not to encompass within this disclosure any previously known products, processes for making products, or methods for using products, and applicants reserve the right to disclaim any previously known products, processes, or methods, which they disclose herein. It is further noted that this disclosure does not intend to encompass within its scope any products, processes, methods for making products, or methods for using products that do not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), and consequently, applicants reserve the right to disclaim any previously described products, processes for making products, or methods for using products, and disclose them herein. Compliance with Art. 53(c) EPC and Rules 28(b) and (c) EPC may be advantageous in the practice of this disclosure. Any right to expressly disclaim any embodiment that is the subject of any granted patent of applicant in this or any other line, or in any prior application of a third party, is expressly reserved. Nothing herein should be construed as a promise.

[0035] It should be noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like can have the meaning ascribed to them in U.S. patent law, and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, e.g., they permit elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of any of the embodiments of the present disclosure.

[0036] These and other embodiments are disclosed or are obvious from and encompassed by the following detailed description. The following detailed description, which is provided by way of example and is not intended to limit any embodiment of the present invention to only the specific embodiments described, can be best understood in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0037] [Figure 1] 1 shows the structural composition of single costimulatory domain receptors and fusion costimulatory domain receptors. A schematic diagram of the claimed CoStAR receptors is shown. The first CoStAR is based on a single costimulatory receptor, and the second fusion CoStAR consists of a full-length costimulatory receptor signaling domain fused to a second costimulatory domain. [Figure 2]Ai-2E: Genomic organization of potential CoStAR constructs—CoStAR consists of an antigen-binding domain, an optional spacer domain, and a costimulatory domain, as shown in the figures and described in the claims. CoStAR can be expressed alone from a promoter (Figures 2Ai-2Aii), with CoStAR consisting of a single (Figure 2Ai) or fusion (Figure 2Aii) costimulatory receptor; (Figure 2B) with an epitope tag (e.g., His tag, DYKDDDDK (SEQ ID NO: 449), etc.) at the N- or C-terminus to allow direct staining of CoStAR; (Figure 2C) with a marker gene separated using a 2A cleavage sequence or an internal ribosome entry site (IRES); (Figure 2D) with a marker gene expressed from a second promoter. (Figure 2E) It may be expressed with a protein of interest, such as a chimeric antigen receptor or T cell receptor, separated using a 2A cleavage sequence or internal ribosome entry site (IRES), or (Figure 2F) it may be expressed with a protein of interest, such as a chimeric antigen receptor or T cell receptor, expressed from a second promoter. It will be apparent to those skilled in the art that CoStAR and the marker gene / chimeric antigen receptor / T cell receptor / other protein of interest can be expressed in either orientation, or 3' (3-prime) or 5' (5-prime) relative to each other. [Figure 3]Figures 3A-3E: Functional activity of CoStAR in T cells in response to LS174T and LoVo tumor-presented antigens. Normal donor T cell populations from donor 1 (Figures 3A and 3D), donor 2 (Figure 3B), and donor 3 (Figures 3C and 3E) were lentivirally engineered to express CoStAR targeting carcinoembryonic antigen (CEA) and magnetically sorted using CD34 magnetic selection to enrich for the transgene. T cells were mixed with wild-type, unmanipulated CEA+ tumor cells (non-activated tumors) or CEA+ tumor cells engineered to express a cell surface-bound anti-CD3 single-chain antibody fragment (activated tumors) at the indicated effector-to-target ratios, and IL-2 was measured in the supernatants by ELISA. Data were obtained using LS174T cells (Figures 3A, 3B, and 3C) and LoVo cells (Figures 3D and 3E). [Figure 4] Figures 4A-4D: Effect of CoStAR on T cell proliferation. 5x10 transduced and untransduced T cells were mixed with 6.25x10 wild-type LoVo or LoVo-OKT3 cells in the presence (Figure 4A) or absence (Figure 4B) of IL-2, and cell counts were performed after 3 days. In separate assays at the same cell ratio, T cells from two donors (Figures 4C and 4D) were loaded with a proliferation dye, and the number of proliferation cycles the cells had undergone was determined by dye dilution using flow cytometry after 6 days. [Figure 5] IL-2 activity of CoStAR fusion receptors in primary human T cells. Normal donor CD8+ T cells from seven donors (except for control CoStAR from three donors) were lentivirally transduced with the indicated CEA-targeted CoStAR, and IL-2 production was assessed after overnight stimulation in the presence of LoVo-OKT3 cells. The proportion of IL-2-positive cells was determined using intracellular flow staining in both CD34-negative (non-CoStAR-transduced) and CD34-positive (CoStAR-transduced) populations. Asterisks indicate significant differences between transduced and non-transduced populations using a paired Wilcoxon signed-rank test, with *p<0.05. [Figure 6A]Multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were retivirally transduced with the indicated CEA-targeted CoStAR, and IL-2 production was assessed after overnight stimulation in the presence of LoVo-OKT3 cells. The percentages of cells positive for IL-2 (seven donors) (Figure 6A), IFNγ (seven donors) (Figure 6B), BCL-xL (five donors) (Figure 6C), and CD107a (six donors) (Figure 6D) were determined using intracellular flow staining in both CD34-negative (non-CoStAR-transduced) and CD34+ (CoStAR-transduced) populations. The control was nonspecific CA125-targeted CoStAR, derived from three donors in all cases. Heatmaps are averages across all donors, and color intensity relates to the percentage of cells positive for a particular readout under the defined conditions. [Figure 6B] Multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were retivirally transduced with the indicated CEA-targeted CoStAR, and IL-2 production was assessed after overnight stimulation in the presence of LoVo-OKT3 cells. The percentages of cells positive for IL-2 (seven donors) (Figure 6A), IFNγ (seven donors) (Figure 6B), BCL-xL (five donors) (Figure 6C), and CD107a (six donors) (Figure 6D) were determined using intracellular flow staining in both CD34-negative (non-CoStAR-transduced) and CD34+ (CoStAR-transduced) populations. The control was nonspecific CA125-targeted CoStAR, derived from three donors in all cases. Heatmaps are averages across all donors, and color intensity relates to the percentage of cells positive for a particular readout under the defined conditions. [Figure 6C]Multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were retivirally transduced with the indicated CEA-targeted CoStAR, and IL-2 production was assessed after overnight stimulation in the presence of LoVo-OKT3 cells. The percentages of cells positive for IL-2 (seven donors) (Figure 6A), IFNγ (seven donors) (Figure 6B), BCL-xL (five donors) (Figure 6C), and CD107a (six donors) (Figure 6D) were determined using intracellular flow staining in both CD34-negative (non-CoStAR-transduced) and CD34+ (CoStAR-transduced) populations. The control was nonspecific CA125-targeted CoStAR, derived from three donors in all cases. Heatmaps are averages across all donors, and color intensity relates to the percentage of cells positive for a particular readout under the defined conditions. [Figure 6D] Multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were retivirally transduced with the indicated CEA-targeted CoStAR, and IL-2 production was assessed after overnight stimulation in the presence of LoVo-OKT3 cells. The percentages of cells positive for IL-2 (seven donors) (Figure 6A), IFNγ (seven donors) (Figure 6B), BCL-xL (five donors) (Figure 6C), and CD107a (six donors) (Figure 6D) were determined using intracellular flow staining in both CD34-negative (non-CoStAR-transduced) and CD34+ (CoStAR-transduced) populations. The control was nonspecific CA125-targeted CoStAR, derived from three donors in all cases. Heatmaps are averages across all donors, and color intensity relates to the percentage of cells positive for a particular readout under the defined conditions. [Figure 7]CD40 enhances IL-2 production from CD28-based CoStAR. Primary human T cells from three healthy donors were either left untransduced or transduced with either extracellular domain-truncated CD28 (TrCD28), full-length CD28 (FLCD28), or CD28.CD40-based CoStAR (MFE23) carrying a CEA-specific scFv. Transduced cells were selected using the CD34 marker gene and expanded prior to analysis. T cells were mixed with OKT3-expressing CEA+ LoVo cells at an effector-to-target ratio of 8:1 for 20 hours, after which IL-2 production was analyzed by ELISA. [Figure 8] Effect of signaling domain and target antigen on CoStAR-mediated T cell expansion. T cells were transduced with either DYKDDDDK (SEQ ID NO: 449) epitope-tagged CD28 or CD28.CD40-based CoStAR bearing CA125-, FolR-, or CEA-specific scFv, or the FolR-specific binding peptide (C7). T cells were mixed with the OKT3-expressing CA125+ / FolR+ / CEA- cell line OVCAR3. The number of transduced cells was counted every 7 days for up to 21 days, and new OVCAR3 cells were added after each count. [Figure 9] Effect of signaling domain and target antigen on CoStAR-mediated T cell expansion. T cells were transduced with either DYKDDDDK (SEQ ID NO: 449) epitope-tagged CD28 or CD28.CD40-based CoStAR bearing CA125-, FolR-, or CEA-specific scFv, or the FolR-specific binding peptide (C7). T cells were mixed with the OKT3-expressing CA125+ / FolR+ / CEA- cell line OVCAR3. The number of transduced cells was counted every 7 days for up to 21 days, and new OVCAR3 cells were added after each count. [Figure 10A](Figures 10A and 10B) CD40-based CoStAR enhances T cell costimulation in a model of TCR transfer. Primary human T cells from three healthy donors were transduced with DYKDDDK-tagged CD28 or CD28.CD40-based CoStAR carrying either MFE (white or black circles) or CA125 (white square)-specific scFvs in addition to a CEA-specific TCR. T cells were mixed with CEA / CA125 H508 cells at a 1:1 effector:target ratio, and intracellular cytokine staining was performed to determine the number of responding CD4 or CD8 T cells in the TCR / CoStAR, TCR / CoStAR, TCR / CoStAR, and TCR / CoStAR populations. Two-way ANOVA (Tukey's test) was performed to determine significant differences in activity: *p>0.05, **p>0.01, ***p>0.001, ****p>0.0001. [Figure 10B] (Figures 10A and 10B) CD40-based CoStAR enhances T cell costimulation in a model of TCR transfer. Primary human T cells from three healthy donors were transduced with DYKDDDK-tagged CD28 or CD28.CD40-based CoStAR carrying either MFE (white or black circles) or CA125 (white square)-specific scFvs in addition to a CEA-specific TCR. T cells were mixed with CEA / CA125 H508 cells at a 1:1 effector:target ratio, and intracellular cytokine staining was performed to determine the number of responding CD4 or CD8 T cells in the TCR / CoStAR, TCR / CoStAR, TCR / CoStAR, and TCR / CoStAR populations. Two-way ANOVA (Tukey's test) was performed to determine significant differences in activity: *p>0.05, **p>0.01, ***p>0.001, ****p>0.0001. [Figure 11]Figure 11 shows the enrichment and expansion of primary human T cells transduced to express the costimulatory molecules of the invention. MFE23 is a single-chain Fv antibody with high affinity for carcinoembryonic antigen (CEA). Primary human T cells were either mock transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 CoStAR, each carrying the CD34 marker gene separated by a 2A cleavage peptide. After in vitro culture, cells were enriched for CD34 using MACS™ paramagnetic selection reagent (Miltenyi Biotech), and then expanded using irradiated feeder cells. An exemplary plot from one of three donors is shown. [Figure 12]Figures 12A-12D show the expansion of T cells transduced with the costimulatory molecules of the present invention in response to stimulation and exogenous IL-2. Cells were mock transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 CoStAR and cocultured with LoVo-OKT3 cells at an effector:target ratio of 8:1 in the presence or absence of exogenous IL-2 (200 IU / ml). On days 1, 4, 7, 11, and 18, cells were harvested and the number of viable T cells was counted using anti-CD2 reagents on a MACSQuant flow cytometer. (Figure 12A) In the absence of tumor and IL-2 stimulation, cell numbers decreased, as expected. (Figure 12B) In the absence of stimulation but in the presence of IL-2, there was more significant cell survival, but no apparent proliferation. (Figure 12C) In the presence of tumor but in the absence of IL-2, mock cells showed no appreciable survival. MFE23.CD28 CoStAR mediated a clear doubling of expansion over the first 4 days followed by a subsequent decline. MFE23.CD28.CD40 mediated a greater expansion up to day 7 followed by a stable decline. (Figure 12D) Under the same conditions but in the presence of IL-2, both mock-transduced and MFE23.CD28-transduced cells showed a 20-fold expansion over 18 days, while MFE23.CD28.CD40 cells expanded more than 60-fold. Thus, CD28.CD40-based receptors exhibit superior expansion and survival under stimulatory conditions, both in the presence and absence of exogenous IL-2. [Figures 13A-13F]Figures 13A-13M show cytokine production by mock-engineered, MFE23.CD28-engineered, or MFE23.CD28.CD40-engineered T cells. Bead array analysis was performed on supernatants obtained from T cell / tumor cocultures. Engineered T cells were incubated with LoVo-OKT3 cells at a 1:1 effector:target ratio for 24 hours, and supernatants were collected. Conditioned supernatants were also collected from equivalent numbers of T cells alone or LoVo-OKT3 cells alone. Cytokine production was analyzed using the Legendplex™ Human TH1 / TH2 Cytokine Panel (Biolegend). (Figure 13A) IL-2, (Figure 13B) IFN-γ, (Figure 13C) TNFα, (Figure 13D) IL-4, (Figure 13E) IL-5, (Figure 13F) IL-13, (Figure 13G) IL-17A, (Figure 13H) IL-17F, (Figure 13I) IL-22, (Figure 13J) IL-6, (Figure 13K) IL-10, (Figure 13L) IL-9, and (Figure 13M) IL-21. Cytokines were either very low or undetectable in media derived from T cells or tumor alone. When cocultured with tumor, cytokine production was enhanced. MFE23.CD28 enhanced the production of IL-2, IL-5, IL-17A / 17F, IL-10, IL-9, and IL-21 compared to mock samples. MFE23.CD28.CD40 also enhanced the production of TNFα, IL-13, and IL-22. MFE23.CD28.CD40 further enhanced the production of some cytokines (IL-2, IL-9, and IL-17F) above that provided by MFE23.CD28, as well as reduced the production of some cytokines (IL-5 and IL-10) below the levels seen with MFE23.CD28. Collectively, this data indicates that adding CD40 to CD28-based costimulatory receptors enhances and / or modulates their specific activity with respect to chemokine production. [Figures 13G-13M]Figures 13A-13M show cytokine production by mock-engineered, MFE23.CD28-engineered, or MFE23.CD28.CD40-engineered T cells. Bead array analysis was performed on supernatants obtained from T cell / tumor cocultures. Engineered T cells were incubated with LoVo-OKT3 cells at a 1:1 effector:target ratio for 24 hours, and supernatants were collected. Conditioned supernatants were also collected from equivalent numbers of T cells alone or LoVo-OKT3 cells alone. Cytokine production was analyzed using the Legendplex™ Human TH1 / TH2 Cytokine Panel (Biolegend). (Figure 13A) IL-2, (Figure 13B) IFN-γ, (Figure 13C) TNFα, (Figure 13D) IL-4, (Figure 13E) IL-5, (Figure 13F) IL-13, (Figure 13G) IL-17A, (Figure 13H) IL-17F, (Figure 13I) IL-22, (Figure 13J) IL-6, (Figure 13K) IL-10, (Figure 13L) IL-9, and (Figure 13M) IL-21. Cytokines were either very low or undetectable in media derived from T cells or tumor alone. When cocultured with tumor, cytokine production was enhanced. MFE23.CD28 enhanced the production of IL-2, IL-5, IL-17A / 17F, IL-10, IL-9, and IL-21 compared to mock samples. MFE23.CD28.CD40 also enhanced the production of TNFα, IL-13, and IL-22. MFE23.CD28.CD40 further enhanced the production of some cytokines (IL-2, IL-9, and IL-17F) above that provided by MFE23.CD28, as well as reduced the production of some cytokines (IL-5 and IL-10) below the levels seen with MFE23.CD28. Collectively, this data indicates that adding CD40 to CD28-based costimulatory receptors enhances and / or modulates their specific activity with respect to chemokine production. [Figures 14A-14F]Figures 14A-14M show the analysis of chemokines using the Legendplex™ human pro-inflammatory chemokine panel: (Figure 14A) IL-8 (CXCL8), (Figure 14B) IP-10 (CSCL10), (Figure 14C) Eotaxin (CCL11), (Figure 14D) TARC (CCL17), (Figure 14E) MCP-1 (CCL2), (Figure 14F) RANTES (CCL5), (Figure 14G) MIP-1a (CCL3), (Figure 14H) MIG (CXCL9), (Figure 14I) ENA-78 (CXCL5), (Figure 14J) MIP-3α (CCL20), (Figure 14K) GROα (CXCL1), (Figure 14L) I-TAC (CXCL11), and (Figure 14M) MEP-1β (CCL4). Chemokine production was either very low or undetectable in medium derived from T cells alone. Chemokine production was enhanced when cocultured with tumor. MFE23.CD28 enhanced the production of CXCL5, CXCL10, CXCL11, CCL17, and CCL20 compared to mock samples. MFE23.CD28.CD40 also enhanced the production of CCL2, CXCL1, and CXCL9. MFE23.CD28.CD40 further enhanced the production of several cytokines (CXCL1, CXCL9, CXCL10, CXCL11, CCL17, CCL2, CXCL9, CCL5, and CCL20) over that provided by MFE23.CD28, as well as reduced the production of some cytokines (CCL4) below the levels seen with MFE23.CD28. Taken together, this data indicates that adding CD40 to CD28-based costimulatory receptors enhances and / or modulates their specific activity with respect to chemokine production. [Figures 14G-14M]Figures 14A-14M show the analysis of chemokines using the Legendplex™ human pro-inflammatory chemokine panel: (Figure 14A) IL-8 (CXCL8), (Figure 14B) IP-10 (CSCL10), (Figure 14C) Eotaxin (CCL11), (Figure 14D) TARC (CCL17), (Figure 14E) MCP-1 (CCL2), (Figure 14F) RANTES (CCL5), (Figure 14G) MIP-1a (CCL3), (Figure 14H) MIG (CXCL9), (Figure 14I) ENA-78 (CXCL5), (Figure 14J) MIP-3α (CCL20), (Figure 14K) GROα (CXCL1), (Figure 14L) I-TAC (CXCL11), and (Figure 14M) MEP-1β (CCL4). Chemokine production was either very low or undetectable in medium derived from T cells alone. Chemokine production was enhanced when cocultured with tumor. MFE23.CD28 enhanced the production of CXCL5, CXCL10, CXCL11, CCL17, and CCL20 compared to mock samples. MFE23.CD28.CD40 also enhanced the production of CCL2, CXCL1, and CXCL9. MFE23.CD28.CD40 further enhanced the production of several cytokines (CXCL1, CXCL9, CXCL10, CXCL11, CCL17, CCL2, CXCL9, CCL5, and CCL20) over that provided by MFE23.CD28, as well as reduced the production of some cytokines (CCL4) below the levels seen with MFE23.CD28. Taken together, this data indicates that adding CD40 to CD28-based costimulatory receptors enhances and / or modulates their specific activity with respect to chemokine production. [Figure 15A]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15B]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15C]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15D]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15E]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15F]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15G]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 15H]Figures 15A-15H show the functional activity of ovarian CoStAR-engineered cells using CoStAR bearing FolR- or CA125-reactive scFvs (MOV19 and 196-14, respectively). Human folate receptor alpha (FolR) represents a preferred target for several tumors, including ovarian, head and neck, kidney, and lung, while CA125 represents an alternative target for ovarian cancer. Primary human T cells derived from six healthy donors were engineered with either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28, or MOV19.CD28.CD40 receptors, all of which carry a DYKDDDDK epitope tag for detection. After mixing the transduced cells with FolR / CA125 OVCAR-OKT3 cells, effector activity was analyzed in the epitope tag-positive and -negative populations using intracellular staining. In CD28- and CD28.CD40-engineered cells, specific enhancement of effector activity, as determined by production of IL-2 (Figures 15A and 15B), TNFα (Figures 15C and 15D), CD137 (Figures 15E and 15F), and BCL-xL (Figures 15G and 15H), was observed in response to both CA125 and FolR, except that specific BCL-xL induction by MOV19.CD28 was not observed compared to MOV19.CD28.CD40. [Figure 16]Figures 16A-16F show three TIL populations that were mock-transduced or engineered with MOV19.CD28.CD40 CoStAR and then mixed with patient-matched tumor digests. Donor tumors displayed varying levels of FolR expression on the digests, ranging from negative (Figure 16A) to low expression (Figure 16B) to high expression (Figure 16C). CoStAR-negative TILs within mock and CoStAR-engineered TIL populations, matched with FolR-negative digests, showed similar levels of CD137 upregulation after tumor coculture, which was not enhanced by the presence of CoStAR (Figure 16D). In TILs exposed to the FolR-low digest, CoStAR+ cells exhibited enhanced activity compared to CoStAR-, with CD137 expression increasing from less than 10% to more than 20% (Figure 16E). In TILs exposed to FolR-rich tumor digest, there was an increase in activity from approximately 20% in the CoStAR- population to approximately 50% in the CoStAR+ population (Figure 16F). [Figure 17A] Figures 17A-17C show enhanced effector function. FolR-targeted CoStAR enhanced CD137 expression from approximately 20% to approximately 50% (Figure 17A), TNFα production from 10% to 15% (Figure 17B), and IL-2 production from 2% to 5% (Figure 17C) in response to FolR+ tumor digests. [Figure 17B] Figures 17A-17C show enhanced effector function. FolR-targeted CoStAR enhanced CD137 expression from approximately 20% to approximately 50% (Figure 17A), TNFα production from 10% to 15% (Figure 17B), and IL-2 production from 2% to 5% (Figure 17C) in response to FolR+ tumor digests. [Figure 17C] Figures 17A-17C show enhanced effector function. FolR-targeted CoStAR enhanced CD137 expression from approximately 20% to approximately 50% (Figure 17A), TNFα production from 10% to 15% (Figure 17B), and IL-2 production from 2% to 5% (Figure 17C) in response to FolR+ tumor digests. [Figure 18]Figures 18A-18F show that soluble ligands do not inhibit effector function. T cells from three healthy donors were engineered with MOV19.CD28 or MOV19.CD28.CD40 CoStAR, provided stimulation in the absence of FolR, and activated with either immobilized OKT3 or OvCAR-OKT3 to obtain TCR and CoStAR activity. BCL-xL activity increased by 10-20% across the three donors after OKT3 stimulation (Figure 18A), whereas IL-2 increased by 0-12% (Figure 18B) and TNFα increased by 0-20% (Figure 18C). The presence of exogenous soluble FolR did not enhance any of these specific effector functions. In the presence of OvCAR-OKT3, BCL-XL induction was enhanced by approximately 20% with CD28 CoStAR but by approximately 35% with CD28.CD40 CoStAR (Fig. 18D), IL-2 induction was enhanced by approximately 20% with CD28 CoStAR but by 30-50% with CD28.CD40 CoStAR (Fig. 18E), and TNFα production was enhanced by 20-30% with CD28 CoStAR but by 25-50% with CD28.CD40 CoStAR (Fig. 18F). Exogenous soluble FolR had no inhibitory effect on any of these effector functions. [Figure 19] Figure 19 shows surface expression of anti-MSLN CoStAR on the surface of HD T cells. Transduced and non-transduced (mock) cells were subjected to a rapid expansion protocol (REP) and assessed for transduction efficiency by either surface detection of the marker gene tCD34 (a truncated form of CD34) using anti-CD34-APC antibodies (black) or surface detection of CoStAR molecules using anti-MSLN-PE antibodies (red). Results represent three biological replicates. [Figure 20]Figures 20A-20C show cytokine expression in healthy donor (HD) T cells transduced with anti-MSLN CoStAR and cocultured with the OVCAR-3 cell line. CoStARs containing six different anti-MSLN scFvs (SS1, M5, HN1, M912, huYP218, and P4) and three different spacer / transmembrane domain combinations (CD28, N-terminally truncated CD28, and CD8) were compared. Structural details are provided in Tables 8, 9, and 10. Cytokine concentrations are shown for (Figure 20A) IL-2, (Figure 20B) IFNγ, and (Figure 20C) TNFα after coculture with the OVCAR-3 or OVCAR3-OKT3 cell line. Untreated T cells were used as controls. Results represent one to three biological replicates, each with three technical replicates. [Figure 21] Figures 21A-21C show cytokine expression in healthy donor (HD) T cells transduced with anti-MSLN CoStAR and cocultured with the K562 cell line. CoStARs containing six different anti-MSLN scFvs (SS1, M5, HN1, M912, huYP218, and P4) and three different spacer / transmembrane domain combinations (CD28, N-terminally truncated CD28, and CD8) were compared. Structural details are provided in Tables 8, 9, and 10. Cytokine concentrations are shown for (Figure 21A) IL-2, (Figure 21B) IFNγ, and (Figure 21C) TNFα after coculture with the K562-MSNL or K562-MSNL-OKT3 cell lines. Untreated T cells were used as controls. Results represent one to three biological replicates, each with three technical replicates. [Figure 22] Figure 22 shows the surface expression of MFE23 scFV anti-CEA CoStAR expressed with six different secretory signal peptides (OSM1, CD8, CD2, IL2, GMCSF, hIgGκ). Structural details are provided in Table 11. After expansion, cells were assessed for transduction efficiency by either surface detection of the marker gene-truncated CD34 (tCD34) using anti-CD34-APC antibody (black bars) or surface detection of CoStAR molecules using primary rhCEACAM5-Fc antibody with secondary anti-IgG-Fc-PE antibody (gray bars), respectively. [Figure 23] Figure 23 shows the surface expression of CoStAR containing six different anti-CEA scFvs (MFE23, MFE23(Q>K), hMFE23, CEA6, BW431 / 26, hT84.66). Structural details are provided in Table 12. After expansion, cells were assessed for transduction efficiency by either surface detection of the marker gene-truncated CD34 (tCD34) using anti-CD34-APC antibody (black bars) or surface detection of CoStAR molecules using primary rhCEACAM5-Fc antibody with secondary anti-IgG-Fc-PE antibody (gray bars). [Figure 24] Figures 24A-24C show cytokine production by healthy donor (HD) T cells transduced with anti-CEA CoStAR (MFE23, MFE23(Q>K), hMFE23, CEA6, BW431 / 26, hT84.66) and co-cultured with LoVo cell line. Structural details are provided in Table 12. Cytokine concentrations after co-culture with Lovo or Lovo-OKT3 cell line are shown for (Figure 24A) IL-2, (Figure 24B) IFNγ, and (Figure 24C) TNFα. Untreated T cells were used as a negative control. [Figure 25] Figures 25A-25C show cytokine production in healthy donor (HD) T cells transduced with anti-CEA CoStAR (MFE23, MFE23(Q>K), hMFE23, CEA6, BW431 / 26, hT84.66) and cocultured with K562 cell lines. Cytokine concentrations after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines are shown for (Figure 25A) IL-2, (Figure 25B) IFNγ, and (Figure 25C) TNFα. Untreated T cells were used as a negative control. [Figure 26]Figure 26 shows the surface expression of hMFE23 scFV anti-CEA CoStAR expressed with three different spacer / transmembrane domains. Structural details are provided in Table 13. After expansion, cells were assessed for transduction efficiency by surface detection of the marker gene truncated CD34 (tCD34) using anti-CD34-APC antibody (black bars) or surface detection of CoStAR molecules using primary rhCEACAM5-Fc antibody with secondary anti-IgG-Fc-PE antibody (gray bars). [Figure 27] Figures 27A-27C show cytokine production by MFE23 scFV anti-CEA spacer variants. Cytokine concentrations are shown for (Figure 27A) IL-2, (Figure 27B) IFNγ, and (Figure 27C) TNFα after coculture with Lovo or Lovo.OKT3 cell lines. Untreated T cells were used as a control. [Figure 28] FIG. 28 shows an anti-CEA CoStAR comprising a hMFE23 CEA binding domain with an intracellular signaling domain containing CD40, CD134, CD137, CD2, ICOS, DAP10, and NTRK1 signaling elements. [Figure 29] Figure 29 shows the surface expression of hMFE23 scFV anti-CEA CoStAR containing the domain combinations shown in Figure 28 and detailed in Table 14. After expansion, cells were assessed for transduction efficiency by surface detection of the marker gene truncated CD34 (tCD34) using anti-CD34-APC antibody (black circles) or CoStAR molecules using primary rhCEACAM5-Fc antibody with secondary anti-IgG-Fc-PE antibody (red circles). Results represent three biological replicates. [Figure 30]Figure 30 shows the T cell phenotype of CoStAR-transfected HD T cells in three separate donors. Cells were sorted using CD34 microbeads and subjected to a 14-day rapid expansion protocol (REP). After expansion and REP, 1 x 10 cells were assessed for differentiation subtypes using flow cytometry. Tcm, central memory T cells; Tem, effector memory T cells; Tn, naive T cells; Tscm, stem cell memory T cells; Tte, terminal effector T cells. See Table 15 for definitions of T cell subtypes. [Figure 31A] Figures 31A-31C show cytokine production by HD T cells transduced with hMFE23 scFV anti-CEA CoStAR and cocultured with K562 cell lines. Cytokine concentrations after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines are shown for (Figure 31A) IL-2, (Figure 31B) IFNγ, and (Figure 31C) TNFα. Untreated T cells were used as a control. [Figure 31B] Figures 31A-31C show cytokine production by HD T cells transduced with hMFE23 scFV anti-CEA CoStAR and cocultured with K562 cell lines. Cytokine concentrations after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines are shown for (Figure 31A) IL-2, (Figure 31B) IFNγ, and (Figure 31C) TNFα. Untreated T cells were used as a control. [Figure 31C] Figures 31A-31C show cytokine production by HD T cells transduced with hMFE23 scFV anti-CEA CoStAR and cocultured with K562 cell lines. Cytokine concentrations after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines are shown for (Figure 31A) IL-2, (Figure 31B) IFNγ, and (Figure 31C) TNFα. Untreated T cells were used as a control. [Figure 32A]Figures 32A-32C show cytokine expression in HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR and cocultured with K562 cell lines. The frequencies of (Figure 32A) IL-2-, (Figure 32B) IFNγ-, and (Figure 32C) TNFα-expressing cells are shown after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines. Untreated T cells were used as a control. [Figure 32B] Figures 32A-32C show cytokine expression in HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR and cocultured with K562 cell lines. The frequencies of (Figure 32A) IL-2-, (Figure 32B) IFNγ-, and (Figure 32C) TNFα-expressing cells are shown after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines. Untreated T cells were used as a control. [Figure 32C] Figures 32A-32C show cytokine expression in HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR and cocultured with K562 cell lines. The frequencies of (Figure 32A) IL-2-, (Figure 32B) IFNγ-, and (Figure 32C) TNFα-expressing cells are shown after coculture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines. Untreated T cells were used as a control. [Figure 33] Figure 33 shows the proliferation of HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR and co-cultured with K562.CEACAM5.OKT3 (signal 1+2) cell line. HD T cells were obtained from two donors. The figure represents the fold expansion of input cells. [Figure 34] Figure 34 shows the proliferation of HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR and co-cultured with the K562.CEACAM5.OKT3 (signal 1+2) cell line. HD T cells were obtained from two donors. Numbers represent fold expansion of input cells at day 6 after stimulation. [Figure 35A]Figures 35A-35B show TRAF and TRAF-like binding sites and motifs and signaling pathways. Figure 35A shows the CoStAR CD40 intracellular domain showing the TRAF binding site and signaling. [Figure 35B] Figures 35A-35B show TRAF and TRAF-like binding sites and motifs and signaling pathways. Figure 35B shows CoStAR containing the IProx domain. [Figure 36A] Figures 36A-36B show the effect of mutations in the CoStAR CD40 intracellular signaling domain on cytokine secretion and long-term survival and proliferation of T cells transduced with CD28.CD40 CoStAR and cocultured with LoVo.OKT3. Cells from three donors were activated with Dynabeads and transduced with wild-type CD28.CD40 (CTP194), CD28.CD40 containing the TRAF2-binding site mutation SVQE>AVQA (CTP195), CD28.CD40 containing the TRAF2 / TRAF3-binding site mutation PVQET>AVAEA (CTP196), CD28.CD40 containing the TRAF6-binding site mutation PQEINF>AQAINF (CTP197), Q263A (CTP199), or mock-transduced. (Figure 36A) IL-2 was measured in supernatants collected 24 hours after coculture with LoVo or LoVo.OKT3.GFP tumor cells in the absence of IL-2. (Figure 36B) After 6–8 days, viability and absolute counts were assessed, and live T cells were re-challenged with fresh LoVo.OKT3.GFP tumor cells for an additional week. At the end of long-term coculture, viability and absolute counts were measured, and fold expansion was calculated. Data are shown as mean ± SEM for n≦3 donors analyzed in triplicate. [Figure 36B]Figures 36A-36B show the effect of mutations in the CoStAR CD40 intracellular signaling domain on cytokine secretion and long-term survival and proliferation of T cells transduced with CD28.CD40 CoStAR and cocultured with LoVo.OKT3. Cells from three donors were activated with Dynabeads and transduced with wild-type CD28.CD40 (CTP194), CD28.CD40 containing the TRAF2-binding site mutation SVQE>AVQA (CTP195), CD28.CD40 containing the TRAF2 / TRAF3-binding site mutation PVQET>AVAEA (CTP196), CD28.CD40 containing the TRAF6-binding site mutation PQEINF>AQAINF (CTP197), Q263A (CTP199), or mock-transduced. (Figure 36A) IL-2 was measured in supernatants collected 24 hours after coculture with LoVo or LoVo.OKT3.GFP tumor cells in the absence of IL-2. (Figure 36B) After 6–8 days, viability and absolute counts were assessed, and live T cells were re-challenged with fresh LoVo.OKT3.GFP tumor cells for an additional week. At the end of long-term coculture, viability and absolute counts were measured, and fold expansion was calculated. Data are shown as mean ± SEM for n≦3 donors analyzed in triplicate. [Figure 37A] 37A-37B show the percentage of TILs based on CD2+ staining in thawed OC samples on day 1 (FIG. 37A). [Figure 37B] Figures 37A-37B show total TIL counts based on CD2+ staining in day 1 thawed OC samples (Figure 37B). [Figure 38] Figure 38 shows proliferation of non-Td and Td TILs. Cells were counted using CD2 and DRAQ7 staining and acquisition on Novocyte 3005. Cell counts at the end of REP on day 25 are depicted graphically. Shapes corresponding to each of the five ovarian cancer samples are shown. Filled shapes are non-Td TILs, and open shapes are Td TILs. Statistical analysis was performed using a paired t-test. [Figure 39A]Figures 39A-39D show the per-cell transduction efficiency and viral integration of CoStAR-modified TILs. Non-transduced (non-Td) TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were assessed for transduction efficiency in CD3+ (Figure 39A), CD4+ (Figure 39B), and CD8+ (Figure 39C) cell populations at day 25 post-REP. Viral copy number (VCN) was assessed to determine viral integration (Figure 39D). Corresponding shapes are shown for each of the five ovarian cancer samples. Filled shapes are non-Td TILs, and open shapes are Td TILs. [Figure 39B] Figures 39A-39D show the per-cell transduction efficiency and viral integration of CoStAR-modified TILs. Non-transduced (non-Td) TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were assessed for transduction efficiency in CD3+ (Figure 39A), CD4+ (Figure 39B), and CD8+ (Figure 39C) cell populations at day 25 post-REP. Viral copy number (VCN) was assessed to determine viral integration (Figure 39D). Corresponding shapes are shown for each of the five ovarian cancer samples. Filled shapes are non-Td TILs, and open shapes are Td TILs. [Figure 39C] Figures 39A-39D show the per-cell transduction efficiency and viral integration of CoStAR-modified TILs. Non-transduced (non-Td) TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were assessed for transduction efficiency in CD3+ (Figure 39A), CD4+ (Figure 39B), and CD8+ (Figure 39C) cell populations at day 25 post-REP. Viral copy number (VCN) was assessed to determine viral integration (Figure 39D). Corresponding shapes are shown for each of the five ovarian cancer samples. Filled shapes are non-Td TILs, and open shapes are Td TILs. [Figure 39D]Figures 39A-39D show the per-cell transduction efficiency and viral integration of CoStAR-modified TILs. Non-transduced (non-Td) TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were assessed for transduction efficiency in CD3+ (Figure 39A), CD4+ (Figure 39B), and CD8+ (Figure 39C) cell populations at day 25 post-REP. Viral copy number (VCN) was assessed to determine viral integration (Figure 39D). Corresponding shapes are shown for each of the five ovarian cancer samples. Filled shapes are non-Td TILs, and open shapes are Td TILs. [Figure 40] Figure 40 shows CD4 and CD8 populations in TILs after REP. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were assessed for CD4 and CD8 composition using flow cytometry on day 25 after REP. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc tests. [Figure 41A] Figures 41A-41C show the effect of CoStAR modification on the differentiation status of TILs. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for their differentiation status from total T cell (Figure 41A), CD4+ (Figure 41B), and CD8+ (Figure 41C) cell populations at 25 days after REP. Statistical analysis was performed using two-way ANOVA with paired Tukey's multiple comparison test. Statistical significance was observed for the CD3+ (Figure 41A) and CD4+ (Figure 41B) populations between anti-FOLR1 CoStAR- Td TILs and anti-FOLR1 CoStAR+ Td TILs for the Tscm population. *p<0.05 [Figure 41B]Figures 41A-41C show the effect of CoStAR modification on the differentiation status of TILs. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for their differentiation status from total T cell (Figure 41A), CD4+ (Figure 41B), and CD8+ (Figure 41C) cell populations at 25 days after REP. Statistical analysis was performed using two-way ANOVA with paired Tukey's multiple comparison test. Statistical significance was observed for the CD3+ (Figure 41A) and CD4+ (Figure 41B) populations between anti-FOLR1 CoStAR- Td TILs and anti-FOLR1 CoStAR+ Td TILs for the Tscm population. *p<0.05 [Figure 41C] Figures 41A-41C show the effect of CoStAR modification on the differentiation status of TILs. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for their differentiation status from total T cell (Figure 41A), CD4+ (Figure 41B), and CD8+ (Figure 41C) cell populations at 25 days after REP. Statistical analysis was performed using two-way ANOVA with paired Tukey's multiple comparison test. Statistical significance was observed for the CD3+ (Figure 41A) and CD4+ (Figure 41B) populations between anti-FOLR1 CoStAR- Td TILs and anti-FOLR1 CoStAR+ Td TILs for the Tscm population. *p<0.05 [Figure 42A]Figures 42A-42B show the effect of CoStAR modification of TILs on the expression of co-inhibitory or co-stimulatory markers. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for the expression of co-inhibitory and co-stimulatory markers in CD4+ (Figure 42A) and CD8+ (Figure 42B) cell populations. Shapes corresponding to each of the five ovarian cancer samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test. *p<0.05 [Figure 42B] Figures 42A-42B show the effect of CoStAR modification of TILs on the expression of co-inhibitory or co-stimulatory markers. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for the expression of co-inhibitory and co-stimulatory markers in CD4+ (Figure 42A) and CD8+ (Figure 42B) cell populations. Shapes corresponding to each of the five ovarian cancer samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test. *p<0.05 [Figure 43A]Figures 43A-43B show the effects of CoStAR modification on TCRαβ, TCRγδ, and Treg frequencies in TILs. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for the frequencies of TCRαβ (CD3+TCRαβ+) and TCRγδ (CD3+TCRγδ+) in the CD3+ cell population (Figure 43A). The frequencies of Tregs in the CD4+ subpopulation were also evaluated (Figure 43B). Shapes corresponding to each of the five ovarian cancer samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test (Figure 43A) and one-way ANOVA with Friedman's post-hoc test (Figure 43B). [Figure 43B] Figures 43A-43B show the effects of CoStAR modification on TCRαβ, TCRγδ, and Treg frequencies in TILs. Non-transduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for the frequencies of TCRαβ (CD3+TCRαβ+) and TCRγδ (CD3+TCRγδ+) in the CD3+ cell population (Figure 43A). The frequencies of Tregs in the CD4+ subpopulation were also evaluated (Figure 43B). Shapes corresponding to each of the five ovarian cancer samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test (Figure 43A) and one-way ANOVA with Friedman's post-hoc test (Figure 43B). [Figure 44A]Figures 44A-44C show the effect of CoStAR modification of TILs on cytokine production upon mitogen activation. Untransduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for cytokine production upon stimulation with PMA (50 ng / mL) / ionomycin (1 μg / mL) for 4 hours in the CD3+ (Figure 44A), CD4+ (Figure 44B), and CD8+ (Figure 44C) cell populations. Shapes corresponding to each of the five OC samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test. *p ≤ 0.05, ***p ≤ 0.001. [Figure 44B] Figures 44A-44C show the effect of CoStAR modification of TILs on cytokine production upon mitogen activation. Untransduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for cytokine production upon stimulation with PMA (50 ng / mL) / ionomycin (1 μg / mL) for 4 hours in the CD3+ (Figure 44A), CD4+ (Figure 44B), and CD8+ (Figure 44C) cell populations. Shapes corresponding to each of the five OC samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test. *p ≤ 0.05, ***p ≤ 0.001. [Figure 44C]Figures 44A-44C show the effect of CoStAR modification of TILs on cytokine production upon mitogen activation. Untransduced (non-Td) TILs, anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+ Td TILs were evaluated for cytokine production upon stimulation with PMA (50 ng / mL) / ionomycin (1 μg / mL) for 4 hours in the CD3+ (Figure 44A), CD4+ (Figure 44B), and CD8+ (Figure 44C) cell populations. Shapes corresponding to each of the five OC samples are shown, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR- Td TILs, and anti-FOLR1 CoStAR+ Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with corresponding Tukey's multiple comparison post-hoc test. *p ≤ 0.05, ***p ≤ 0.001. [Figure 45] Figure 45 shows the expression of FOLR1 in OC digest cells. Cryopreserved autologous tumor digests were thawed and analyzed by flow cytometry to determine surface expression of FOLR1 for each donor. All five donors used in this study are shown. Abbreviations: OC, ovarian cancer; FOLR1, folate receptor alpha; FOLR1 PE, anti-FOLR1 antibody conjugated to PE; PE, phycoerythrin. [Figure 46] Figures 46A-46D show the effect of CoStAR modification on cytokine-producing cells during coculture with autologous tumors. Non-Td TILs and anti-FOLR1 CoStAR Td TILs were cocultured with autologous tumor digests for 16 hours, and intracellular flow cytometry was performed to assess the proportion of cytokine-producing cells. The frequencies of IL-2-positive (Figure 46A) CD4 and (Figure 46B) CD8 TILs and TNFα-positive (Figure 46C) CD4 and (Figure 46D) CD8 TILs were assessed. Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *P<0.05. [Figure 47]Figures 47A-47E show the effect of CoStAR modification on cytokine secretion during coculture with autologous tumor digests expressing FOLR1. Non-Td TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were cocultured with autologous tumor digests for 24 hours, after which supernatants were collected and analyzed for cytokine secretion using an MSD immunoassay. Graphs represent the measured concentrations of (Figure 47A) IL-2, (Figure 47B) TNFα, (Figure 47C) IL-13, and (Figure 47D) IFNγ in the coculture supernatants. (Figure 47E) Correlation between IFNγ concentration and FOLR1 expression by autologous digests, as determined in Figure 1. Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Sidak's multiple comparison test. Correlation analysis was performed using simple linear regression. *P<0.05, **P<0.01, ***P<0.001. [Figure 48] Figure 48 shows the evaluation of MHC-dependent CoStAR functionality in cocultures with autologous tumor digests. Non-Td TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were cocultured with autologous tumor digests for 24 hours in the presence of MHC-blocking reagent or isotype control. Supernatants were then analyzed for IFNγ cytokine production using an MSD immunoassay. Results represent three biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. [Figure 49]Figures 49A-49B show the effect of CoStAR modification on the frequency of cytokine-producing cells during coculture with engineered cell lines. Non-Td TILs and anti-FORL1 CoStAR-transduced (Td) TILs were cocultured with engineered target cell lines for 16 hours, and cytokine-producing cells were measured using intracellular flow cytometry. The frequency of IL-2-positive CD4+ (top) and CD8+ (bottom) TILs after coculture with K-562 (left) and OVCAR-3 (right) derived lines (Figure 49A). The frequency of TNFα-positive CD4+ (top) and CD8+ (bottom) TILs after coculture with K-562 (left) and OVCAR-3 (right) derived lines (Figure 49B). Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 50] Figures 50A-50D show the effect of CoStAR modification on cytokine secretion during coculture with engineered cell lines. Non-Td TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were cocultured with engineered target cell lines for 24 hours, and MSD immunoassays were performed to assess secreted cytokine concentrations. Cytokine concentrations are shown for (Figure 50A) IL-2, (Figure 50B) TNFα, (Figure 50C) IL-13, and (Figure 50D) IFNγ after coculture with (left) K-562- and (right) BA / F3-derived cell lines. Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 51A]Figures 51A-51C show an evaluation of the impact of CoStAR modification on the cytotoxic capacity of TILs against OKT3-bearing targets. Non-Td TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were cocultured with BA / F3- or OVCAR-3-engineered cell lines, and cytotoxicity was assessed using a flow cytometry-based assay (Figure 51A) and an xCELLigence-based assay (Figures 51B and 51C), respectively. (Figure 51B) An E:T ratio of 1:5 and (Figure 51C) An E:T ratio of 1:30. Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test for flow cytometry data and one-way ANOVA with Tukey's multiple comparison test for xCELLigence data. *P<0.05, **P<0.01. [Figure 51B] Figures 51A-51C show an evaluation of the impact of CoStAR modification on the cytotoxic capacity of TILs against OKT3-bearing targets. Non-Td TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were cocultured with BA / F3- or OVCAR-3-engineered cell lines, and cytotoxicity was assessed using a flow cytometry-based assay (Figure 51A) and an xCELLigence-based assay (Figures 51B and 51C), respectively. (Figure 51B) An E:T ratio of 1:5 and (Figure 51C) An E:T ratio of 1:30. Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test for flow cytometry data and one-way ANOVA with Tukey's multiple comparison test for xCELLigence data. *P<0.05, **P<0.01. [Figure 51C]Figures 51A-51C show an evaluation of the impact of CoStAR modification on the cytotoxic capacity of TILs against OKT3-bearing targets. Non-Td TILs and anti-FOLR1 CoStAR-transduced (Td) TILs were cocultured with BA / F3- or OVCAR-3-engineered cell lines, and cytotoxicity was assessed using a flow cytometry-based assay (Figure 51A) and an xCELLigence-based assay (Figures 51B and 51C), respectively. (Figure 51B) An E:T ratio of 1:5 and (Figure 51C) An E:T ratio of 1:30. Results represent five biological replicates with three technical replicates each. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test for flow cytometry data and one-way ANOVA with Tukey's multiple comparison test for xCELLigence data. *P<0.05, **P<0.01. [Figure 52] Figures 52A-52D show expansion of NSCLC TILs with >80% viability (Figure 52A) and approximately 100-200 million cells (Figure 52B), 40-55% CoStar transduction in CD3+ T cells (Figure 52C), and >90% CD3+ T cell purity (Figure 52D). [Figure 53] Figure 53 shows the expansion of NSCLC TILs with a survival rate of over 80%. There was no difference in survival rate and cell expansion between CoStAR-transduced and non-transduced populations. [Figure 54] Figure 54 shows the expansion of NSCLC TILs, which were harvested at 50-500 million cells. There was no difference in viability or cell expansion between the CoStAR-transduced and non-transduced populations. [Figure 55] Figure 55 shows the T cell populations of seven patient samples. [Figure 56] Figure 56 shows CD3 versus CD56, with CD3+ marking T cells and CD3-CD56+ marking NK cells. [Figure 57] Figure 57 shows the quantification of untransduced and CoStAR-transduced T cells. [Figure 58] Figure 58 shows CoStAR staining in T cell subsets. [Figure 59] Figures 59A-59D show a significant increase in ovarian cancer TIL viability during REP and greater than 90% viability at the end of the manufacturing process, with 100-300 million cells at harvest. Figure 59A shows cell counts of ovarian cancer TILs over 10 days, and Figure 59B shows viability. Figure 59C shows cell counts after the manufacturing process, and Figure 59D shows viability. [Figure 60] Figures 60A-60D show that kidney cancer TILs consistently showed greater than 50% viability during expansion and greater than 90% viability at the end of the manufacturing process, with 100-350 million cells at harvest. Figure 60A shows cell counts of kidney cancer TILs over 10 days, and Figure 60B shows viability. Figure 60C shows cell counts after the manufacturing process, and Figure 60D shows viability. [Figure 61] Figure 61 shows the experimental setup outlined in Example 16 in which CD40 signaling motif mutant constructs were introduced into cells to screen for changes to transcription. [Figure 62A] FIG. 62A shows the 23-day experimental approach for producing the TRAF variant CoSTAR in T cells, as outlined in Example 17. [Figure 62B] FIG. 62B shows the 26-day experimental approach to monitoring the effect of the TRAF variant CoSTAR on proliferation in T cells, as outlined in Example 17. [Figure 63] Figures 63A-63C show the change in total CD2+ T cell numbers expressing the TRAF variant CoSTAR with IL-2 treatment. The graphs show the change in cell numbers obtained from cells collected from donors HD 702C (Figure 63A), HD 1004 (Figure 63B), and HD 2000 (Figure 63C). [Figure 64]Figures 64A-64C show the fold change in the number of CD2+ T cells expressing the TRAF variant CoSTAR with IL-2 treatment. The graphs show the fold change in cell number obtained from cells collected from donors HD 702C (Figure 64A), HD 1004 (Figure 64B), and HD 2000 (Figure 64C). [Figure 65] Figures 65A-65C show the change in total CD2+ T cell numbers expressing the TRAF variant CoSTAR when T cells did not receive any IL-2 treatment. The graphs show the change in cell numbers obtained from cells collected from donors HD 702C (Figure 65A), HD 1004 (Figure 65B), and HD 2000 (Figure 65C). [Figure 66] Figures 66A-66C show the fold change in CD2+ T cell numbers expressing the TRAF variant CoSTAR when T cells did not receive any IL-2 treatment. The graphs show the fold change in cell numbers obtained from cells collected from donors HD 702C (Figure 66A), HD 1004 (Figure 66B), and HD 2000 (Figure 66C). [Figure 67-1] Figure 67 shows several embodiments of CD40 TRAF variants of SEQ ID NOS: 510-519. In this figure, the underlined region is CD40, the sequence highlighted in gray is the TRAF6 binding domain region, the sequence highlighted in light gray is the TRAF2 / 3 binding domain, and the sequence highlighted in gray and enclosed in double brackets is the TRAF2 binding domain. [Figure 67-2] Figure 67-1 continued. [Figure 67-3] Figure 67-2 continued. [Figure 68] Figure 68 shows the transduction levels of constructs into primary T cells collected from donors HD 702C, HD 1004, and HD 2000. [Figure 69A]Figures 69A-69F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 69A and 69D), 1004 (Figures 69B and 69E), and 2000 (Figures 69C and 69F) expressing CoStAr constructs with TRAF6 variants either with (Figures 69A-69C) or without (Figures 69D-69F) IL-2 treatment, as outlined in Example 18. [Figure 69B] Figures 69A-69F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 69A and 69D), 1004 (Figures 69B and 69E), and 2000 (Figures 69C and 69F) expressing CoStAr constructs with TRAF6 variants either with (Figures 69A-69C) or without (Figures 69D-69F) IL-2 treatment, as outlined in Example 18. [Figure 69C] Figures 69A-69F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 69A and 69D), 1004 (Figures 69B and 69E), and 2000 (Figures 69C and 69F) expressing CoStAr constructs with TRAF6 variants either with (Figures 69A-69C) or without (Figures 69D-69F) IL-2 treatment, as outlined in Example 18. [Figure 69D] Figures 69A-69F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 69A and 69D), 1004 (Figures 69B and 69E), and 2000 (Figures 69C and 69F) expressing CoStAr constructs with TRAF6 variants either with (Figures 69A-69C) or without (Figures 69D-69F) IL-2 treatment, as outlined in Example 18. [Figure 69E]Figures 69A-69F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 69A and 69D), 1004 (Figures 69B and 69E), and 2000 (Figures 69C and 69F) expressing CoStAr constructs with TRAF6 variants either with (Figures 69A-69C) or without (Figures 69D-69F) IL-2 treatment, as outlined in Example 18. [Figure 69F] Figures 69A-69F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 69A and 69D), 1004 (Figures 69B and 69E), and 2000 (Figures 69C and 69F) expressing CoStAr constructs with TRAF6 variants either with (Figures 69A-69C) or without (Figures 69D-69F) IL-2 treatment, as outlined in Example 18. [Figure 70A] Figures 70A-70F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 70A and 70D), 1004 (Figures 70B and 70E), and 2000 (Figures 70C and 70F) expressing CoStAr constructs with TRAF2 / 3 variants, either with (Figures 70A-70C) or without (Figures 70D-70F) IL-2 treatment, as outlined in Example 18. [Figure 70B] Figures 70A-70F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 70A and 70D), 1004 (Figures 70B and 70E), and 2000 (Figures 70C and 70F) expressing CoStAr constructs with TRAF2 / 3 variants, either with (Figures 70A-70C) or without (Figures 70D-70F) IL-2 treatment, as outlined in Example 18. [Figure 70C]Figures 70A-70F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 70A and 70D), 1004 (Figures 70B and 70E), and 2000 (Figures 70C and 70F) expressing CoStAr constructs with TRAF2 / 3 variants, either with (Figures 70A-70C) or without (Figures 70D-70F) IL-2 treatment, as outlined in Example 18. [Figure 70D] Figures 70A-70F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 70A and 70D), 1004 (Figures 70B and 70E), and 2000 (Figures 70C and 70F) expressing CoStAr constructs with TRAF2 / 3 variants, either with (Figures 70A-70C) or without (Figures 70D-70F) IL-2 treatment, as outlined in Example 18. [Figure 70E] Figures 70A-70F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 70A and 70D), 1004 (Figures 70B and 70E), and 2000 (Figures 70C and 70F) expressing CoStAr constructs with TRAF2 / 3 variants, either with (Figures 70A-70C) or without (Figures 70D-70F) IL-2 treatment, as outlined in Example 18. [Figure 70F] Figures 70A-70F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 70A and 70D), 1004 (Figures 70B and 70E), and 2000 (Figures 70C and 70F) expressing CoStAr constructs with TRAF2 / 3 variants, either with (Figures 70A-70C) or without (Figures 70D-70F) IL-2 treatment, as outlined in Example 18. [Figure 71A]Figures 71A-71F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 71A and 71D), 1004 (Figures 71B and 71E), and 2000 (Figures 71C and 71F) expressing CoStAr constructs with TRAF2 variants either with (Figures 71A-71C) or without (Figures 71D-71F) IL-2 treatment, as outlined in Example 18. [Figure 71B] Figures 71A-71F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 71A and 71D), 1004 (Figures 71B and 71E), and 2000 (Figures 71C and 71F) expressing CoStAr constructs with TRAF2 variants either with (Figures 71A-71C) or without (Figures 71D-71F) IL-2 treatment, as outlined in Example 18. [Figure 71C] Figures 71A-71F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 71A and 71D), 1004 (Figures 71B and 71E), and 2000 (Figures 71C and 71F) expressing CoStAr constructs with TRAF2 variants either with (Figures 71A-71C) or without (Figures 71D-71F) IL-2 treatment, as outlined in Example 18. [Figure 71D] Figures 71A-71F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 71A and 71D), 1004 (Figures 71B and 71E), and 2000 (Figures 71C and 71F) expressing CoStAr constructs with TRAF2 variants either with (Figures 71A-71C) or without (Figures 71D-71F) IL-2 treatment, as outlined in Example 18. [Figure 71E]Figures 71A-71F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 71A and 71D), 1004 (Figures 71B and 71E), and 2000 (Figures 71C and 71F) expressing CoStAr constructs with TRAF2 variants either with (Figures 71A-71C) or without (Figures 71D-71F) IL-2 treatment, as outlined in Example 18. [Figure 71F] Figures 71A-71F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 71A and 71D), 1004 (Figures 71B and 71E), and 2000 (Figures 71C and 71F) expressing CoStAr constructs with TRAF2 variants either with (Figures 71A-71C) or without (Figures 71D-71F) IL-2 treatment, as outlined in Example 18. [Figure 72A] Figures 72A-72F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 72A and 72D), 1004 (Figures 72B and 72E), and 2000 (Figures 72C and 72F) expressing CoStAr constructs with various TRAF variants, either with (Figures 72A-72C) or without (Figures 72D-72F) IL-2 treatment, as outlined in Example 18. [Figure 72B] Figures 72A-72F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 72A and 72D), 1004 (Figures 72B and 72E), and 2000 (Figures 72C and 72F) expressing CoStAr constructs with various TRAF variants, either with (Figures 72A-72C) or without (Figures 72D-72F) IL-2 treatment, as outlined in Example 18. [Figure 72C]Figures 72A-72F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 72A and 72D), 1004 (Figures 72B and 72E), and 2000 (Figures 72C and 72F) expressing CoStAr constructs with various TRAF variants, either with (Figures 72A-72C) or without (Figures 72D-72F) IL-2 treatment, as outlined in Example 18. [Figure 72D] Figures 72A-72F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 72A and 72D), 1004 (Figures 72B and 72E), and 2000 (Figures 72C and 72F) expressing CoStAr constructs with various TRAF variants, either with (Figures 72A-72C) or without (Figures 72D-72F) IL-2 treatment, as outlined in Example 18. [Figure 72E] Figures 72A-72F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 72A and 72D), 1004 (Figures 72B and 72E), and 2000 (Figures 72C and 72F) expressing CoStAr constructs with various TRAF variants, either with (Figures 72A-72C) or without (Figures 72D-72F) IL-2 treatment, as outlined in Example 18. [Figure 72F] Figures 72A-72F show the fold change in cell proliferation compared to baseline in primary T cells from primary human donors 702C (Figures 72A and 72D), 1004 (Figures 72B and 72E), and 2000 (Figures 72C and 72F) expressing CoStAr constructs with various TRAF variants, either with (Figures 72A-72C) or without (Figures 72D-72F) IL-2 treatment, as outlined in Example 18. [Figure 73A]Figures 73A-73D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells treated with IL-2 and represent the combined data for cells expressing TRAF6 (Figure 73A), TRAF2 / 3 (Figure 73B), TRAF2 (Figure 73C), or other TRAF (Figure 73D) variants. [Figure 73B] Figures 73A-73D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells treated with IL-2 and represent the combined data for cells expressing TRAF6 (Figure 73A), TRAF2 / 3 (Figure 73B), TRAF2 (Figure 73C), or other TRAF (Figure 73D) variants. [Figure 73C] Figures 73A-73D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells treated with IL-2 and represent the combined data for cells expressing TRAF6 (Figure 73A), TRAF2 / 3 (Figure 73B), TRAF2 (Figure 73C), or other TRAF (Figure 73D) variants. [Figure 73D] Figures 73A-73D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells treated with IL-2 and represent the combined data for cells expressing TRAF6 (Figure 73A), TRAF2 / 3 (Figure 73B), TRAF2 (Figure 73C), or other TRAF (Figure 73D) variants. [Figure 74A]Figures 74A-74D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells without IL-2 treatment and represent the combined data for cells expressing TRAF6 (Figure 74A), TRAF2 / 3 (Figure 74B), TRAF2 (Figure 74C), or other TRAF (Figure 74D) variants. [Figure 74B] Figures 74A-74D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells without IL-2 treatment and represent the combined data for cells expressing TRAF6 (Figure 74A), TRAF2 / 3 (Figure 74B), TRAF2 (Figure 74C), or other TRAF (Figure 74D) variants. [Figure 74C] Figures 74A-74D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells without IL-2 treatment and represent the combined data for cells expressing TRAF6 (Figure 74A), TRAF2 / 3 (Figure 74B), TRAF2 (Figure 74C), or other TRAF (Figure 74D) variants. [Fig. 74D] Figures 74A-74D show the combined data from Figures 69A-69C, 70A-70C, 71A-71C, and 72A-72C, graphing the fold change in cell proliferation relative to baseline over time in primary T cells across three donors. The graphs show cells without IL-2 treatment and represent the combined data for cells expressing TRAF6 (Figure 74A), TRAF2 / 3 (Figure 74B), TRAF2 (Figure 74C), or other TRAF (Figure 74D) variants. [Figure 75A]Figures 75A-75B show the combined fold change in cell proliferation compared to baseline in primary human T cells from three donors at day 21 for cells with (Figure 75A) and without (Figure 75B) IL-2 treatment. [Figure 75B] Figures 75A-75B show the combined fold change in cell proliferation compared to baseline in primary human T cells from three donors at day 21 for cells with (Figure 75A) and without (Figure 75B) IL-2 treatment. [Figure 76A] Figures 76A-76C show non-limiting examples of schematic models of universal costimulatory proteins with TRAF variants. (Figure 76A) A TCR-incorporated antigen agnostic receptor (TIAAR) contains modifications of components of the TCR complex and associated signaling adaptors. (Figure 76B) A constitutive costimulatory receptor contains a transmembrane domain (TMD) and features that allow for inducible or constitutive activation. (Figure 76C) An inducible costimulatory receptor can be induced and activated by extracellular ligand binding. In this non-limiting example, the TRAF variant is part of the CD40 domain (shown as "CD40*"). [Figure 76B] Figures 76A-76C show non-limiting examples of schematic models of universal costimulatory proteins with TRAF variants. (Figure 76A) A TCR-incorporated antigen agnostic receptor (TIAAR) contains modifications of components of the TCR complex and associated signaling adaptors. (Figure 76B) A constitutive costimulatory receptor contains a transmembrane domain (TMD) and features that allow for inducible or constitutive activation. (Figure 76C) An inducible costimulatory receptor can be induced and activated by extracellular ligand binding. In this non-limiting example, the TRAF variant is part of the CD40 domain (shown as "CD40*"). [Figure 76C]Figures 76A-76C show non-limiting examples of schematic models of universal costimulatory proteins with TRAF variants. (Figure 76A) A TCR-incorporated antigen agnostic receptor (TIAAR) contains modifications of components of the TCR complex and associated signaling adaptors. (Figure 76B) A constitutive costimulatory receptor contains a transmembrane domain (TMD) and features that allow for inducible or constitutive activation. (Figure 76C) An inducible costimulatory receptor can be induced and activated by extracellular ligand binding. In this non-limiting example, the TRAF variant is part of the CD40 domain (shown as "CD40*"). [Figure 77] Figures 77A-77C show non-limiting exemplary protein constructs of some embodiments herein. Figure 77A is a schematic diagram of a protein comprising a universal CoStAR sequence and further comprising an optional compartment, a binding domain, a CD28 domain, and a CD40 domain with a TRAF variant. Figure 77B is a schematic diagram of some embodiments provided herein. Figure 77C outlines a set of sequences of some embodiments provided herein. [Figure 78] Figure 78 shows a set of sequences (SEQ ID NOS: 40839-41074) of some embodiments provided herein. In some embodiments, these sequences are excluded from the consensus sequences or other TRAF variant constructs provided herein (including those in Figures 84 and 85). [Figure 79] Figure 79 shows a set of sequences (SEQ ID NOS: 41075-43474) according to some embodiments provided herein. In some embodiments, these sequences are excluded from the consensus sequences or other TRAF variant constructs provided herein (including those in Figures 84 and 85). [Figure 80] FIG. 80 shows a non-limiting example of a mutational analysis of CD40 using constructs CTP195, CTP196, and CTP197 to assess the function of signaling motifs, as described in Example 13. [Figure 81] FIG. 81 shows a non-limiting example of mutational analysis of CD40 using constructs CTP195, CTP196, CTP197, CTP198, and CTP199 to assess the function of signaling motifs, as described in Example 13. [Figure 82A] Figure 82A shows the minor consensus sequence generated from the alignment of variants in the TRAF2 domain shown in Table 1, with highlighted amino acids conserved across the variants. [Figure 82B] Figure 82B shows the major consensus sequence generated from the alignment of variants in the TRAF2 domain shown in Table 1, with highlighted amino acids conserved across the variants. [Figure 82C] Figure 82C shows the consensus sequence generated from the alignment of variants in the TRAF6 domain shown in Table 3, with highlighted amino acids conserved across the variants. [Figure 83] Figure 83 shows non-limiting exemplary sequences of variants in the TRAF2 domain (SEQ ID NOs: 520-679). [Figure 84] Figure 84 shows non-limiting exemplary sequences of variants in the TRAF2 / 3 domain (SEQ ID NOs: 680-839). [Figure 85] Figure 85 shows non-limiting exemplary sequences of variants in the TRAF6 domain (SEQ ID NOs: 840-40839). [Figures 86A-86C]Figures 86A-86D are schematic diagrams showing several embodiments of FOLR1-alpha CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 86A. Figure 86B shows several embodiments of FOLR1-alpha CoStAR or fusion proteins. Figure 86C shows several embodiments of CoStAR or fusion proteins. Figure 86D shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figure 86D]Figures 86A-86D are schematic diagrams showing several embodiments of FOLR1-alpha CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 86A. Figure 86B shows several embodiments of FOLR1-alpha CoStAR or fusion proteins. Figure 86C shows several embodiments of CoStAR or fusion proteins. Figure 86D shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figures 87A-87C]Figures 87A-87D are schematic diagrams showing several embodiments of anti-pembrolizumab CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 87A. Figure 87B shows several embodiments of anti-pembrolizumab CoStAR or fusion proteins. Figure 87C shows several embodiments of CoStAR or fusion proteins. Figure 87D shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figure 87D]Figures 87A-87D are schematic diagrams showing several embodiments of anti-pembrolizumab CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 87A. Figure 87B shows several embodiments of anti-pembrolizumab CoStAR or fusion proteins. Figure 87C shows several embodiments of CoStAR or fusion proteins. Figure 87D shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figures 88A-88C]Figures 88A-88D are schematic diagrams showing several embodiments of CEA CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 88A. Figure 88B shows several embodiments of CEA CoStAR or fusion proteins. Figure 88C shows several embodiments of CoStAR or fusion proteins. Figure 88D shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figure 88D]Figures 88A-88D are schematic diagrams showing several embodiments of CEA CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 88A. Figure 88B shows several embodiments of CEA CoStAR or fusion proteins. Figure 88C shows several embodiments of CoStAR or fusion proteins. Figure 88D shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figures 89A-89C]Figures 89A-89D are schematic diagrams showing several embodiments of CEA CoStAR and / or fusion proteins with CD40 variants. Several general embodiments are shown in Figure 89A. Figure 89B shows several embodiments of MSLN CoStAR or fusion proteins. Figure 89C shows several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figure 89D-1] Figures 89A-89D are schematic diagrams illustrating several embodiments of CEA CoStAR and / or fusion proteins with CD40 variants. Figure 89D illustrates several embodiments of CoStAR or fusion proteins. In some embodiments, the sequence describes the structure in its entirety, and no additional functional aspects are required to describe the CoStAR or fusion protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. [Figure 89D-2] Figure 89-1 continued. [Figure 89D-3] Figure 89-2 continued. [Figure 89D-4] Figure 89-3 continued. [Figure 90A] 90A-90C show the amount of cytokine TNF-alpha (FIG. 90A) present after development of T cells bearing CD40 variants, as outlined in Example 19. [Figure 90B] 90A-90C show the amount of IFN-gamma (FIG. 90B) present after expression of T cells bearing CD40 variants, as outlined in Example 19. [Figure 90C] 90A-90C show the amount of cytokine IL-2 (FIG. 90C) present after development of T cells bearing CD40 variants, as outlined in Example 19. [Figure 91A] 91A-91B show IL-2 production in cells expressing one of the constructs CTP194-CTP200 (FIG. 91A), as outlined in Example 20. [Figure 91B] 91A-91B show the fold expansion in cells expressing one of the constructs CTP194-CTP200 (FIG. 91B), as outlined in Example 20. [Figure 92] FIG. 92 shows a schematic diagram of the protein constructs CTP188-CTP202 described herein. DETAILED DESCRIPTION OF THE INVENTION

[0038] The disclosure herein relates to sequences encoding variants of TRAF-binding domains. In some embodiments, these variants may be of the TRAF2, TRAF2 / 3, or TRAF6 domains. In some embodiments, a TRAF2 variant will comprise one or more of the variants or point mutations provided herein. In some embodiments, a TRAF2 / 3 variant will comprise one or more of the variants or point mutations provided herein. In some embodiments, a TRAF6 variant will comprise one or more of the variants or point mutations provided herein. In some embodiments, the TRAF2, TRAF2 / 3, or TRAF6 variant will be any one or more of those described herein, including, for example, one or more of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519. In some embodiments, the variant will comprise any one or more of the constructs from Table 2, Table 4, Table 5, or Figure 67. In some embodiments, the variant is not one of the constructs in Figure 78 (SEQ ID NOs: 40839-41074) or Figure 79 (SEQ ID NOs: 41075-43474). In some embodiments, the variant comprises at least one of the constructs in Figure 83, Figure 84, or Figure 85. In some embodiments, the TRAF2, TRAF2 / 3, or TRAF6 variant will be part of a CD40 protein. In some embodiments, the CD40 protein is part of a CoSTAR protein or other fusion construct provided herein. In some embodiments, the TRAF variant is expressed in a T cell provided herein. In some embodiments, the TRAF variant is expressed in a cell as shown in Figures 76A-76C. In some embodiments, the TRAF variant is part of a peptide construct as shown in Figures 77A-77C, 86A-86D, 87A-87D, 88A-88D, and / or 89A-89D.

[0039] In some embodiments, any of the proteins and / or CoSTARs provided herein may utilize a CD40 or TRAF variant in place of the wild-type CD40 or TRAF domain. Any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, as well as SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. With respect to any description of CD40, TRAF, TRAF2, TRAF2 / 3, and TRAF6 variants provided herein, in some embodiments, the sequences in Figures 78 and 79 may be excluded from the variant options of the present invention.

[0040] In some embodiments, the sequence is a variant of TRAF2. In some embodiments, the sequence is selected from any one of SEQ ID NOs: 520-679. In some embodiments, the sequence has about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to any one of SEQ ID NOs: 520-679. In some embodiments, the sequence is a variant of TRAF2 / 3. In some embodiments, the sequence is selected from any one of SEQ ID NOs: 680-839. In some embodiments, the sequence has about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to any one of SEQ ID NOs: 680-839. In some embodiments, the sequence is a variant of TRAF6. In some embodiments, the sequence is selected from any one of SEQ ID NOs: 840-40839. In some embodiments, the sequence has about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60 and about 100% identity to any one of SEQ ID NOs: 840-40839. In some embodiments, the sequence includes at least two, three, and / or four variants of TRAF.

[0041] In some embodiments, the sequence comprises a sequence from Table 2, 4, or 5. In some embodiments, the sequence comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60 and about 100% identity to a sequence from Table 2, 4, or 5. In some embodiments, the sequence is selected from any one of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, and 519. In some embodiments, the sequence has about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to any one of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, and 519.

[0042] Some embodiments of the present disclosure include sequences of consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 In some embodiments, X1 is any amino acid, X2 is any amino acid, X3 is P, X4 is any amino acid, X5 is Q, X6 is C, E, or A, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid, and X 10 is any amino acid, and X 11 In some embodiments, X1 and X2 are any amino acid, X3 is P, X4 is any amino acid, X5 is Q, and X6, X7, X8, X9, X 10 , and X 11 is any amino acid. In some embodiments, X1 to X 11 Any one of X1 to X2 is a naturally occurring amino acid. 11In some embodiments, any one of X1 and X2 is an altered amino acid. In some embodiments, X3 and / or X5 are conservatively mutated to another amino acid with similar properties to P and / or Q, respectively. In some embodiments, the protein is part of a TRAF domain. In some embodiments, the protein is part of a TRAF2 domain. In some embodiments, the protein is part of a TRAF2 / 3 domain. In some embodiments, the sequences are: X1 is S or P; X2 is V, H, or Y; X4 is I, Q, V; X7 is T or S; X8 is D; X9 is K, D, or G; and X 10 is T, S, G, or A, and X 11 is D, S, N, or D, or the sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or at least 100% identical to (a). In some embodiments, the protein comprises the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42299. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42377. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42378.

[0043] In some embodiments, the sequence of consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, S, A, or T, X4 is any amino acid, X5 is Q or E, X6 is E, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid, and X 10is any amino acid, and X 11 In some embodiments, a protein is provided, wherein X1 to X are any amino acid. 11 Any one of X1 to X2 is a naturally occurring amino acid. 11 In some embodiments, any one of X3 is an altered amino acid. In some embodiments, X3 is conservatively mutated to another amino acid with similar properties to P, S, A, and / or T. In some embodiments, X5 is conservatively mutated to another amino acid with similar properties to Q and / or E. In some embodiments, X6 is conservatively mutated to another amino acid with similar properties to E. In some embodiments, the protein is part of a TRAF domain. In some embodiments, the protein is part of a TRAF2 domain. In some embodiments, the protein is part of a TRAF2 / 3 domain. In some embodiments, the sequence is: X1 is P, A, M, T, S, R, V, H; X2 is F, A, L, I, T, V, G, C, A, F, P; X4 is K, V, I, H, A, Q, T, E, S; X7 is C, T, E, D, S, A; X8 is A, L, G, Y, I, Q, D, E; X9 is F, H, K, R, P, A, G, Y, E, N; 10 are R, G, E, K, A, S, D, C, C, P, V, and X 11 is S, C, D, P, W, T, A, G, E, or the sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or at least 100% identical to (a). In some embodiments, the sequence is a portion of the Traf2 / 3 sequence. In some embodiments, the protein comprises the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42300. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11QPVT, SEQ ID NO: 42379. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42380.

[0044] In some embodiments, a protein is provided comprising a consensus C sequence or an amino acid sequence comprising X1X2X3X4X5X6, wherein X1 is P, X2 is any amino acid, X3 is E, X4 is any amino acid, X5 is any amino acid, and X6 is Ac / Ar. In some embodiments, X1 is P, and X2, X3, X4, X5, and X6 are any amino acids. In some embodiments, any one of X1-X6 is a naturally occurring amino acid. In some embodiments, any one of X1-X6 is a modified amino acid. In some embodiments, X1 is conservatively mutated to another amino acid with properties similar to P. In some embodiments, X6 is an aromatic amino acid. In some embodiments, X6 is an acidic amino acid. In some embodiments, X6 is conservatively mutated to another amino acid with properties similar to acidic and / or aromatic amino acids. In some embodiments, X6 is selected from a Y, H, W, or F amino acid. In some embodiments, X6 is selected from a D or E amino acid. In some embodiments, the protein is a portion of a TRAF domain. In some embodiments, the protein is a portion of a TRAF6 domain. In some embodiments, the sequence comprises a sequence where X2 is Q, P, E, V, T, or S, X4 is I, L, M, N, S, T, N, D, and X5 is N, D, R, S, G, or Y, or the sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or at least 100% identical to (a). In some embodiments, the protein comprises the sequence PKQEX1X2X3X4X5X6X7X8X9X 10 X 11, SEQ ID NO: 42301. In some embodiments, the protein is of the sequence X1X2X3X4X5X6PDDL, SEQ ID NO: 42381. In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0045] Consensus sequences A, B, and C were determined as shown in Tables 1 and 3 and Figures 82A-82C. In some embodiments, the amino acid sequence comprises (a) one or more of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519, or (b) a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or at least 100% identical to (a).

[0046] In some embodiments, the TRAF domain is in a CD40 protein, wherein the TRAF domain comprises one or more of: (a) SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; or (b) a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or at least 100% identical to (a).

[0047] In some embodiments, the TRAF6 domain sequence comprises PQEINF, PEEMSW, PPENYE, or PQENSY. In some embodiments, the TRAF2 / 3 domain sequence comprises PVQET, TQEET, SKEET, AVEET, or PVQET. In some embodiments, the TRAF2 domain sequence comprises SVQE, AVEE, or PEEE.

[0048] In some embodiments, the TRAF6 or TRAF2 / 3 domain comprises (a) the amino acid sequence of one of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519, or (b) a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or at least 100% identical to (a). In some embodiments, the sequence is within the CD40 protein sequence for the protein, amino acid sequence, or TRAF domain of any one of the embodiments of the present disclosure. In some embodiments, the sequence is within the CD40 protein and is located in the corresponding TRAF2, TRAF2 / 3, and / or TRAF6 domain within the CD40 protein.

[0049] In some embodiments, the TRAF2 / TRAF3 domain has consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, X4 is any amino acid, X5 is Q, X6 is C, E, or A, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid, and X 10 is any amino acid, and X 11 is any amino acid. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42299. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42377. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42378.

[0050] In some embodiments, the TRAF2 / TRAF3 domain has consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, S, A, or T, X4 is any amino acid, X5 is Q or E, X6 is E, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid, and X 10 is any amino acid, and X 11 is any amino acid. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 , SEQ ID NO: 42300. In some embodiments, the protein has the sequence X1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, SEQ ID NO: 42379. In some embodiments, the protein has the sequence GSNTX1X2X3X4X5X6X7X8X9X 10 X 11 QPVT, sequence number 42380. In some embodiments, the TRAF6 domain comprises the consensus C or X1X2X3X4X5X6 amino acid sequence, where X1 is P, X2 is any amino acid, X3 is E, X4 is any amino acid, X5 is any amino acid, and X6 is Ac / Ar. In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6, SEQ ID NO: 42301. In some embodiments, the protein is of the sequence X1X2X3X4X5X6PDDL, SEQ ID NO: 42381. In some embodiments, the protein is of the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0051] In some embodiments, the CD40 domain comprises one or more of the following amino acid sequences: (a) consensus A or X1X2X3X4X5X6X7X8X9X10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, X4 is any amino acid, X5 is Q, X6 is C, E, or A, X7 is any amino acid, X8 is any amino acid, and X9 is any amino acid; 10 is any amino acid, and X 11 is any amino acid; (b) a TRAF2 / TRAF3 domain having consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 wherein X1 is any amino acid, X2 is any amino acid, X3 is P, S, A, or T, X4 is any amino acid, X5 is Q or E, X6 is E, X7 is any amino acid, X8 is any amino acid, and X9 is any amino acid; 10 is any amino acid, and X 11 is any amino acid; (c) a TRAF6 domain comprising the amino acid sequence of consensus C or X1X2X3X4X5X6, where X1 is P, X2 is any amino acid, X3 is E, X4 is any amino acid, X5 is any amino acid, and X6 is Ac / Ar.

[0052] In some embodiments, a chimeric costimulatory antigen receptor (CoStAR) is provided, comprising: an extracellular binding domain that binds to a tumor-associated antigen; a transmembrane domain linked to the extracellular binding domain; a first signaling domain; and a second signaling domain, wherein the second signaling domain comprises a variant CD40 signaling domain or a signaling fragment thereof, wherein the variant CD40 comprises a variant TRAF2 / TRAF3 sequence, or a variant TRAF6 sequence, or a variant TRAF2 sequence, and wherein the variant CD40 does not comprise SEQ ID NO: 42. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0053] In some embodiments, a chimeric costimulatory antigen receptor (CoStAR) is provided, comprising: an extracellular binding domain that binds to carcinoembryonic antigen (CEA) or an extracellular binding domain that binds to mesothelin (MSLN); a transmembrane domain linked to the extracellular binding domain; a first signaling domain; and a second signaling domain, wherein the second signaling domain comprises a variant CD40 signaling domain or a signaling fragment thereof, wherein the variant CD40 comprises a variant TRAF2 / TRAF3 sequence, a variant TRAF6 sequence, or a variant TRAF2 sequence, and wherein the variant CD40 does not comprise SEQ ID NO: 42. In some embodiments, the first signaling domain comprises a signaling domain or a signaling fragment of CD28 or CD278 (ICOS). In some embodiments, the first signaling domain comprises a full-length costimulatory domain. In some embodiments, the construct is as shown in Figure 88A. In some embodiments, the construct is as shown in Figure 88B. In some embodiments, the construct is as shown in Figure 88C. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0054] In some embodiments, the extracellular binding domain is operably linked to the transmembrane domain via a linker and / or spacer. In some embodiments, the linker comprises about 2, 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, or any integer between 2 and 350 amino acids. In some embodiments, the linker comprises about 5 to about 20 amino acids. In some embodiments, the linker comprises about 10 to about 250 amino acids. In some embodiments, the linker comprises 5 amino acids. In some embodiments, the linker comprises 10 amino acids. In some embodiments, the linker comprises 20 amino acids. In some embodiments, the linker comprises 50 amino acids. In some embodiments, the linker comprises 100 amino acids. In some embodiments, the linker comprises 250 amino acids.

[0055] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from CD28, CD8, ICOS, DAP10, or NTRK. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from CD28. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from ICOS. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from DAP10. In some embodiments, the transmembrane domain comprises a transmembrane domain sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the transmembrane domain comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to any one of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the extracellular binding domain comprises an scFv, a peptide, an antibody heavy chain variable domain, an antibody light chain variable domain, or a CEA ligand. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and Tables 2, 4-5, and 7-14, as well as SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0056] In some embodiments, the nucleic acid encodes a CD40 TRAF variant construct of any one of the embodiments of the present disclosure. In some embodiments, the nucleic acid encodes CoStAR of any one of the embodiments of the present disclosure. In some embodiments, the vector comprises a nucleic acid of any one of the embodiments of the present disclosure.

[0057] In some embodiments, the cell expresses a CD40 TRAF variant construct of any one of the embodiments of the present disclosure. In some embodiments, the cell comprises an alpha-beta T cell, a gamma-delta T cell, a regulatory T cell, a TIL, an NKT cell, or an NK cell. In some embodiments, the cell co-expresses a CAR or a TCR. In some embodiments, the cell is a cell that expresses any one of the CoStAR embodiments of the present disclosure. In some embodiments, the cell comprises an alpha-beta T cell, a gamma-delta T cell, a regulatory T cell, a TIL, an NKT cell, or an NK cell. In some embodiments, the cell co-expresses a CAR or a TCR.

[0058] In some embodiments, a method of making a cell of any one of the embodiments of the present disclosure, the method comprising transducing or transfecting the cell with a vector of any one of the embodiments of the present disclosure. In some embodiments, a method for preparing a cell population expressing CoStAR of any one of the embodiments of the present disclosure, comprising detecting expression of CoStAR on the surface of cells transfected or transduced with the vector of Alternative Method 62, and selecting cells identified as expressing CoStAR. In some embodiments, the cell population is enriched for cellular expression of CoStAR of any one of the embodiments of the present disclosure. In some embodiments, a method for treating a disease in a subject, the method comprising administering to the subject a cell according to any one of the embodiments of the present disclosure or a cell population according to any one of the embodiments of the present disclosure.

[0059] In some embodiments, the protein comprises the amino acid sequence of one of SEQ ID NOs: 510-519. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to any one of SEQ ID NOs: 510-519. In some embodiments, the protein comprises the sequence of SEQ ID NO: 510. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 510. In some embodiments, the protein comprises the sequence of SEQ ID NO: 511. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 511. In some embodiments, the protein comprises a sequence of SEQ ID NO: 512. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 512. In some embodiments, the protein comprises a sequence of SEQ ID NO: 513. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 513. In some embodiments, the protein comprises a sequence of SEQ ID NO: 514. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 514. In some embodiments, the protein comprises a sequence of SEQ ID NO: 515. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 515.In some embodiments, the protein comprises the sequence of SEQ ID NO: 516. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 516. In some embodiments, the protein comprises the sequence of SEQ ID NO: 517. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 517. In some embodiments, the protein comprises the sequence of SEQ ID NO: 518. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identity to SEQ ID NO: 518. In some embodiments, the protein comprises the sequence of SEQ ID NO: 519. In some embodiments, the protein comprises a sequence having about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60 and about 100% identity to SEQ ID NO: 519. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. The sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0060] In some embodiments, the immune cell comprises an extracellular tumor-associated antigen (TAA)-specific binding domain and a transmembrane domain, the immune cell comprising a) a CD28 intracellular signaling domain and b) a CD40 intracellular signaling domain, wherein the CD40 intracellular signaling domain comprises a TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the immune cell is an isolated nucleic acid molecule encoding a chimeric receptor for an immune cell comprising an extracellular tumor-associated antigen (TAA)-specific binding domain and a transmembrane domain, the nucleic acid molecule further comprising a TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0061] In some embodiments, the present invention is a chimeric costimulatory receptor (CoStAR) comprising an extracellular ligand-binding fragment of a first costimulatory receptor and an intracellular signaling fragment of a second costimulatory receptor fused to a tumor-associated antigen-specific binding domain linked to a signaling domain, wherein the CoStAR further comprises a TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the CoStAR comprises an antigen-specific binding domain selected from (a) a single-chain antibody fragment that binds to a tumor-associated antigen, including, but not limited to, carcinoembryonic antigen (CEA), 5T4, melanotransferrin (CD228), Her2, EGFR, GPC3, melanoma-associated chondroitin sulfate proteoglycan (MCSP / CSPG4), CD71, folate receptor, or CA125; or (b) a single-chain antibody fragment that binds to a tumor-specific peptide (p)-major histocompatibility (MHO) complex; or (c) a tumor-specific pMHC complex antigen-specific single-chain T cell receptor (scTCR); or (d) a naturally occurring antigen-binding polypeptide, such as, but not limited to, transferrin; or (e) an antibody (e.g., but not limited to, an Fe-binding domain, e.g., CD16, CD32, or CD64), or other indirect methods of antigen recognition. In some embodiments, CoStAr comprises (1) a fusion signaling domain comprising or consisting of full-length human CD28, or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60-100% sequence identity at the protein level; (2) an intracellular domain derived from human CD137, e.g., or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60-100% sequence identity at the protein level; (3) an intracellular domain derived from human CD134, or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60-100% sequence identity at the protein level; (4) an intracellular domain derived from human CD2, or a variant thereof having at least 60%, 70%,(5) an intracellular domain derived from human CD29 or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60 and 100% sequence identity at the protein level; (6) an intracellular domain derived from human GITR or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60 and 100% sequence identity at the protein level; (7) an intracellular domain derived from human IL2R-gamma or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60 and 100% sequence identity at the protein level. (8) an intracellular domain derived from human CD40 or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60 and 100% sequence identity at the protein level; (9) an intracellular domain derived from human CD150 or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60 and 100% sequence identity at the protein level; and / or (10) an intracellular domain derived from human CD2 and human CD40 or a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100%, or any integer between 60 and 100% sequence identity at the protein level. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins.May be excluded from all of the embodiments provided herein.

[0062] In some embodiments, the present invention is a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain operably linked to a transmembrane domain, a CD28 signaling domain, and a CD40 signaling domain, wherein at least one extracellular binding domain comprises the amino acid sequence of SEQ ID NO: 42275, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 42276, and the CD40 signaling domain comprises the amino acid sequence of SEQ ID NO: 42277. In some embodiments, the CoStAR further comprises at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the present invention is a protein comprising a first portion comprising the amino acid sequence of SEQ ID NO: 42275 linked to a second portion comprising a transmembrane domain, said second portion linked to a third portion comprising the amino acid sequence of SEQ ID NO: 42276, and said third portion linked to a fourth portion comprising the amino acid sequence of SEQ ID NO: 42277. In some embodiments, the protein further comprises at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the protein comprises a first portion comprising the amino acid sequence of SEQ ID NO: 42275, a second portion comprising the amino acid sequence of SEQ ID NO: 42278, a third portion comprising the amino acid sequence of SEQ ID NO: 42276, a fourth portion comprising the amino acid sequence of SEQ ID NO: 42277, and a fifth portion comprising SEQ ID NO: 42279, wherein the first portion is linked to the second portion by the fifth portion, the third portion is linked to the second portion, and the fourth portion is linked to the third portion. In some embodiments, the protein further comprises at least one TRAF variant of any one of the embodiments of the present disclosure.In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0063] In some embodiments, the present invention is a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain comprising the VH of antibody 196-14 and the VL of antibody 196-14 operably linked to a transmembrane domain, a CD28 signaling domain, and a CD40 signaling domain, wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 42276 and the CD40 signaling domain comprises the amino acid sequence of SEQ ID NO: 42277. In some embodiments, the CoStAR further comprises at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the cell population is enriched for cellular expression of CoStAR, wherein CoStAR comprises an extracellular binding domain comprising the VH of antibody 196-14 and the VL of antibody 196-14 operably linked to a transmembrane domain, a CD28 signaling domain, and a CD40 signaling domain, wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 42276, and the CD40 signaling domain comprises the amino acid sequence of SEQ ID NO: 42277. In some embodiments, the CoStAR further comprises at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0064] In some embodiments, the CoStAR comprises an extracellular binding domain operably linked to a transmembrane domain, a first signaling domain, a CD40 signaling domain or a fragment thereof, and at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the CD40 signaling fragment comprises an SH3 motif (KPTNKAPH, SEQ ID NO: 42280), a TRAF2 motif (PKQE, SEQ ID NO: 42281, PVQE, SEQ ID NO: 42282, SVQE, SEQ ID NO: 42283), a TRAF6 motif (QEPQEINFP, SEQ ID NO: 42284), a PKA motif (KKPTNKA, SEQ ID NO: 42285, SRISVQE, SEQ ID NO: 42286), or a combination thereof, or is the full-length CD40 intracellular domain. In some embodiments, the first signaling domain comprises a signaling domain or signaling fragment of CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. In some embodiments, the extracellular binding domain binds to CD70, CD146, FOLRI, carcinoembryonic antigen (CEA), 5T4, melanotransferrin (CD228), Her2, EGFR, GPC3, melanoma-associated chondroitin sulfate proteoglycan (MCSP / CSPG4), CD71, EPCAM, SM5-1, folate receptor, or CA125, PDL-1, CD155, PD-I, mesothelin, or a tumor-specific peptide (p)-major histocompatibility (MHC) complex, or a tumor-specific pMHC complex antigen-specific single-chain T-cell receptor (scTCR), or transferrin, or an antibody or antigen-binding protein.In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0065] In some embodiments, the receptor is a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain specific for a tumor-associated antigen (TAA) operably linked to a transmembrane domain, a CD28 signaling domain, a CD40 signaling domain or a signaling fragment thereof, and at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the CoStAR is specific for FOLR1. In some embodiments, the CoStAR is specific for PDL1. In some embodiments, the CoStAR is specific for CEA. In some embodiments, the extracellular binding domain comprises a MOV19 scFv. In some embodiments, the extracellular binding domain is specific for CD70, CD146, FOLR1, carcinoembryonic antigen (CEA), 5T4, melanotransferrin (CD228), Her2, EGFR, GPC3, melanoma-associated chondroitin sulfate proteoglycan (MCSP / CSPG4), CD71, EPCAM, SM5-1, folate receptor or CA125, PDL-1, CD155, PD-1, mesothelin, or a tumor-specific peptide (p)-major histocompatibility (MHC) complex, or a tumor-specific pMHC complex antigen-specific single-chain T-cell receptor (scTCR), or transferrin, or an antibody or antigen-binding protein. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0066] In some embodiments, the fusion protein comprises at least one TRAF variant of any one of the embodiments of this disclosure, (a) a binding domain comprising an L-CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 42287, an L-CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 42288, an L-CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 42289, an H-CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 42290, an H-CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 42291, and an H-CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 173, (b) the amino acid sequence of SEQ ID NO: 42293 linked to the binding domain, (c) the amino acid sequence of SEQ ID NO: 42294 linked to (b), (d) the amino acid sequence of SEQ ID NO: 42295 linked to (c), and (e) the amino acid sequence of SEQ ID NO: 42296 linked to (d). In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 42297 or 42298, and at least one TRAF variant of any one of the embodiments of this disclosure. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0067] In some embodiments, the engineered protein comprises a clustering domain, and a signaling domain comprising a CD40 signaling domain or a signaling fragment thereof, and at least one TRAF variant of any one of the embodiments of the present disclosure, wherein the clustering domain is capable of oligomerization, thereby activating the signaling domain. In some embodiments, the protein comprises a constitutively stimulating antigen agnostic receptor (C-SAAR). In some embodiments, the protein comprises at least one of LZ(cFos)-EGFR™ / JMD-CD28-CD40, LZ(cFos)-CD28™-CD28-CD40, LZ(cJun)-EGFR™ / JMD-CD28-CD40, LZ(cJun)-CD28™-CD28-CD40, LZ(c / EBP)-EGFR™ / JMD-CD28-CD40, or LZ(c / EBP)-CD28™-CD28-CD40. In some embodiments, the protein further comprises an inducible costimulatory receptor. In some embodiments, the protein comprises at least one of anti-ID1-VHVL(A3 0514-pembro)-CD28TMD-CD28-CD40, or anti-ID1-VL-VH(A3 0514-pembro)CD28TMD-CD28-CD40, or anti-ID2-VH-VL(A3 0523-pembro)-CD28TMD-CD28-CD40, or anti-ID2-VL-Vh(A3 0523-pembro)-CD28TMD-CD28-CD40, or anti-ID3-Vh-VL(A3063 3-pembro)-CD28TMD-CD28-CD40, or anti-ID3-VL-VH(A 3063 3-pembro)-CD28TMD-CD28-CD40. In some embodiments, the engineered protein comprises a transmembrane domain and the clustering domain oligomerizes when the engineered protein is subjected to binding of a ligand.In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0068] In some embodiments, the CoStAR comprises an extracellular binding domain that binds to carcinoembryonic antigen (CEA) and at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the extracellular binding domain is operably linked to ICOS, wherein ICOS comprises the amino acid sequence of SEQ ID NO: 515, wherein ICOS is linked to a CD40 signaling domain, and wherein the extracellular binding domain comprises an HCDR1 that is the HCDR1 in SEQ ID NO: 12, an HCDR2 that is the HCDR2 in SEQ ID NO: 12, an HCDR3 that is the HCDR3 in SEQ ID NO: 12, an LCDR1 that is the LCDR1 in SEQ ID NO: 12, an LCDR2 that is the LCDR2 in SEQ ID NO: 12, and an LCDR3 that is the HCDR3 in SEQ ID NO: 12. In some embodiments, a fusion protein comprising at least one TRAF variant of any one of the embodiments of the present disclosure, further comprising an HCDR1 that is the HCDR1 in SEQ ID NO: 12, an HCDR2 that is the HCDR2 in SEQ ID NO: 12, an HCDR3 that is the HCDR3 in SEQ ID NO: 12, an LCDR1 that is the LCDR1 in SEQ ID NO: 12, an LCDR2 that is the LCDR2 in SEQ ID NO: 12, and an LCDR3 that is the HCDR3 in SEQ ID NO: 12. In some embodiments, the construct is as shown in Figure 88A. In some embodiments, the construct is as shown in Figure 88B. In some embodiments, the construct is as shown in Figure 88C. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0069] In some embodiments, a CoStAR comprises an extracellular binding domain that binds to mesothelin (MSLN) and at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, CoStAR further comprises a transmembrane domain, a CD28 signaling domain comprising the amino acid sequence of SEQ ID NO: 25, a CD40 signaling domain, and the extracellular binding domain is selected from the group consisting of HCDR1 which is HCDR1 in SEQ ID NO: 186, HCDR2 which is HCDR2 in SEQ ID NO: 186, HCDR3 which is HCDR3 in SEQ ID NO: 186, LCDR1 which is LCDR1 in SEQ ID NO: 186, LCDR2 which is LCDR2 in SEQ ID NO: 186, and LCDR3 which is LCDR3 in SEQ ID NO: 186, HCDR1 which is HCDR1 in SEQ ID NO: 187, HCDR2 which is HCDR2 in SEQ ID NO: 187, HCDR3 which is HCDR3 in SEQ ID NO: 187, LCDR1 which is LCDR1 in SEQ ID NO: 187, LCDR2 which is LCDR2 in SEQ ID NO: 187, and LCDR3 which is LCDR3 in SEQ ID NO: 187, HCDR1 which is HCDR1 in SEQ ID NO: 188, HCDR2 which is HCDR2 in SEQ ID NO: 188, SEQ ID NO: HCDR3 which is HCDR3 in SEQ ID NO: 188, LCDR1 which is LCDR1 in SEQ ID NO: 188, LCDR2 which is LCDR2 in SEQ ID NO: 188, and LCDR3 which is LCDR3 in SEQ ID NO: 188, HCDR1 which is HCDR1 in SEQ ID NO: 189, HCDR2 which is HCDR2 in SEQ ID NO: 189, HCDR3 which is HCDR3 in SEQ ID NO: 189, LCDR1 which is LCDR1 in SEQ ID NO: 189, LCDR2 which is LCDR2 in SEQ ID NO: 189, and LCDR3 which is LCDR3 in SEQ ID NO: 189, HCDR1 which is HCDR1 in SEQ ID NO: 190, HCDR2 which is HCDR2 in SEQ ID NO: 190, HCDR3 which is HCDR3 in SEQ ID NO: 190, LCDR1 which is LCDR1 in SEQ ID NO: 190, LCDR2 which is LCDR2 in SEQ ID NO: 190, and LCDR3 which is LCDR3 in SEQ ID NO: 190, HCDR1 which is HCDR1 in SEQ ID NO: 191, HCDR2 which is HCDR2 in SEQ ID NO: 191,and an LCDR3 that is the HCDR3 in SEQ ID NO: 191, an LCDR1 that is the LCDR1 in SEQ ID NO: 191, an LCDR2 that is the LCDR2 in SEQ ID NO: 191, and an LCDR3 that is the LCDR3 in SEQ ID NO: 191, a VH that is the VH in SEQ ID NO: 186 and a VL that is the VL in SEQ ID NO: 186, a VH that is the VH in SEQ ID NO: 187 and a VL that is the VL in SEQ ID NO: 187, a VH that is the VH in SEQ ID NO: 188 and a VL that is the VL in SEQ ID NO: 188, a VH that is the VH in SEQ ID NO: 189 and a VL that is the VL in SEQ ID NO: 189, a VH that is the VH in SEQ ID NO: 190 and a VL that is the VL in SEQ ID NO: 190, or a VH that is the VH in SEQ ID NO: 191 and a VL that is the VL in SEQ ID NO: 191. In some embodiments, a fusion protein comprising at least one TRAF variant of any one of the embodiments of the present disclosure. In some embodiments, the fusion protein comprises HCDR1 which is HCDR1 in SEQ ID NO: 186, HCDR2 which is HCDR2 in SEQ ID NO: 186, HCDR3 which is HCDR3 in SEQ ID NO: 186, LCDR1 which is LCDR1 in SEQ ID NO: 186, LCDR2 which is LCDR2 in SEQ ID NO: 186, and LCDR3 which is LCDR3 in SEQ ID NO: 186, HCDR1 which is HCDR1 in SEQ ID NO: 187, HCDR2 which is HCDR2 in SEQ ID NO: 187, HCDR3 which is HCDR3 in SEQ ID NO: 187, LCDR1 which is LCDR1 in SEQ ID NO: 187, LCDR2 which is LCDR2 in SEQ ID NO: 187 LCDR2 is R2, and LCDR3 is LCDR3 in SEQ ID NO: 187, HCDR1 is HCDR1 in SEQ ID NO: 188, HCDR2 is HCDR2 in SEQ ID NO: 188, HCDR3 is HCDR3 in SEQ ID NO: 188, LCDR1 is LCDR1 in SEQ ID NO: 188, LCDR2 is LCDR2 in SEQ ID NO: 188, and LCDR3 is LCDR3 in SEQ ID NO: 188, HCDR1 is HCDR1 in SEQ ID NO: 189, HCDR2 is HCDR2 in SEQ ID NO: 189, HCDR3 is HCDR3 in SEQ ID NO: 189, LCDR1 is LCDR1 in SEQ ID NO: 189,LCDR2 is LCDR2 in SEQ ID NO: 189, and LCDR3 is LCDR3 in SEQ ID NO: 189, HCDR1 is HCDR1 in SEQ ID NO: 190, HCDR2 is HCDR2 in SEQ ID NO: 190, HCDR3 is HCDR3 in SEQ ID NO: 190, LCDR1 is LCDR1 in SEQ ID NO: 190, LCDR2 is LCDR2 in SEQ ID NO: 190, and LCDR3 is LCDR3 in SEQ ID NO: 190, HCDR1 is HCDR1 in SEQ ID NO: 191, HCDR2 is HCDR2 in SEQ ID NO: 191, HCDR3 is HCDR3 in SEQ ID NO: 191, LCDR1 is LCDR1 in SEQ ID NO: 191 1 and LCDR3 that is LCDR3 in SEQ ID NO: 191; a VH that is the VH of SEQ ID NO: 186 and a VL that is the VL of SEQ ID NO: 186; a VH that is the VH of SEQ ID NO: 187 and a VL that is the VL of SEQ ID NO: 187; a VH that is the VH of SEQ ID NO: 188 and a VL that is the VL of SEQ ID NO: 188; a VH that is the VH of SEQ ID NO: 189 and a VL that is the VL of SEQ ID NO: 189; a VH that is the VH of SEQ ID NO: 190 and a VL that is the VL of SEQ ID NO: 190; or a VH that is the VH of SEQ ID NO: 191 and a VL that is the VL of SEQ ID NO: 191. In some embodiments, the fusion protein further comprises a second sequence that is a transmembrane domain, a third sequence that comprises the amino acid sequence of SEQ ID NO: 25, and a fourth sequence that comprises the amino acid sequence of SEQ ID NO: 32, wherein the first sequence is linked to the second sequence, the second sequence is linked to the third sequence, and the third sequence is linked to the fourth sequence. In some embodiments, the fusion protein further comprises a CD40, TRAF2, TRAF2 / 3, or TRAF6 variant provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C.The sequences in Figures 78 and 79 may be omitted from all of the embodiments provided herein.

[0070] In some embodiments, the present disclosure relates to a CoStAR comprising at least one TRAF variant of any one of the embodiments of the present disclosure, wherein the CoStAR is capable of CoStAR ligand-induced activation of a T cell receptor (TCR)-bearing immune cell when the TCR is engaged by a target of the TCR, and the CoStAR comprises an extracellular ligand-binding domain specific for the inducible ligand, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the present disclosure relates to a cell expressing the CoStAR, antibody, or protein of any one of the embodiments of the present disclosure. In some embodiments, the cell comprises an alpha-beta T cell. In some embodiments, the alpha-beta T cell comprises a tumor-infiltrating lymphocyte (TIL). In some embodiments, the CoStAR does not comprise a full-length CD28 extracellular domain. In some embodiments, the CoStAR comprises a truncated CD28 extracellular domain or a CD8 extracellular domain located between the ligand-binding domain and the transmembrane domain. In some embodiments, the intracellular signaling domain comprises one or more of CD28, CD40, CD137, OX40, ICOS, CD2, or DAP10, or signaling fragments thereof. In some embodiments, the ligand-binding domain binds to tafasitamab, cetuximab, trastuzumab, and / or pembrolizumab. In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (excluding those in Figures 78 and 79), and Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C, can be used in the methods and / or constructs and / or proteins. The sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0071] In some embodiments, a method of cell therapy includes identifying a subject in need thereof and administering a CoStAR, fusion protein, nucleic acid, vector, or cell of any one of the embodiments of the present disclosure. In some embodiments, a method for preparing a cell population enriched in CoStAR or protein expressing a TRAF variant, comprising detecting expression of CoStAR or protein on the surface of cells transfected or transduced with a vector of any one of the embodiments of the present disclosure, and selecting cells identified as expressing CoStAR or protein. In some embodiments, a method for treating a disease in a subject in need thereof, comprising administering to the subject a cell or cell population of any one of the embodiments of the present disclosure.

[0072] CD40 and TRAF CD40 is a member of the TNF receptor superfamily that provides activation signals in antigen-presenting cells, such as B cells, macrophages, and dendritic cells. Multimerization of CD40 with its ligand initiates signal transduction by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. The TRAF-binding site is responsible for the intracellular signal transduction of CD40.

[0073] The TRAF domain mediates the oligomerization of TRAF proteins and their association with upstream receptors or adaptors and downstream effector proteins. Within the large family of E3 ubiquitinase ligases, the RING domain is best known for its function in mediating protein ubiquitination. Among the major downstream pathways regulated by TRAFs are those that result in the activation of nuclear factor kappa beta (NF-κB) and mitogen-activated protein kinases (MAPKs), which are important for the induction of genes associated with innate immunity, inflammation, and cell survival. TRAF2 and TRAF3 function as negative regulators of several signaling pathways involved in B cell survival and the inflammatory response of innate immune cells.

[0074] The canonical pathway of NF-κB is induced by TNFα, IL-1, or LPS and involves IKK activation using diverse signaling adaptors. Phosphorylation of serine residues in the signal-responsive region (SRR) of canonical IκB by IKKβ triggers IκB ubiquitination and subsequent proteasomal degradation. This results in the release of NF-κB dimers, which can then translocate to the nucleus and induce target gene transcription. The noncanonical pathway relies on NIK (NF-κB-inducing kinase)-induced activation of IKKα. IKKα phosphorylates the p100 NF-κB subunit, which leads to proteasomal processing of p100 to p52. This leads to activation of p52-RelB dimers, which target specific κB elements.

[0075] It has been discovered that costimulatory receptors containing a CD40 signaling domain exhibit novel and improved activity profiles. It has further been discovered that variants of the TRAF2, TRAF2 / 3, and TRAF6-binding domains within CD40 modulate signal transduction. The activity profile can be modulated by selecting an intracellular domain of the receptor protein that binds to the CD40 signaling domain and / or by selecting an element of the CD40 signaling domain to bind to the intracellular domain of the receptor protein. Provided herein is a recombinant costimulatory antigen receptor (CoStAR) comprising (i) a disease- or tumor-associated antigen-binding domain, (ii) a first intracellular segment comprising the intracellular signaling domain of the receptor protein, and (iii) a second intracellular signaling domain of the CD40 receptor protein or a signaling fragment thereof. The CoStAR may comprise an extracellular segment of a stimulatory receptor protein. In some embodiments, the extracellular segment of the stimulatory receptor protein is capable of binding to a ligand. In some embodiments, the extracellular segment of the stimulatory receptor protein is truncated and does not bind to a ligand. In some embodiments, the extracellular segment of the stimulatory receptor protein acts as a spacer of adjustable length, allowing the disease- or tumor-associated antigen-binding domain to be positioned away from the surface of the cell in which it is expressed, for example, to form a more optimal immune synapse. In some embodiments, the extracellular segment of the stimulatory receptor protein and the first intracellular segment comprise segments of the same receptor protein. In some embodiments, the extracellular segment and the first intracellular segment comprise segments of different receptor proteins. CoStAR comprises a transmembrane domain interposed between the disease- or tumor-associated antigen-binding domain and the first intracellular domain. If the extracellular segment of the stimulatory receptor protein is present, the transmembrane domain is interposed between the extracellular segment and the first intracellular signaling domain.In some embodiments, any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C. Sequence options in Figures 78 and 79 may be excluded from all of the embodiments provided herein.

[0076] As used herein, "full-length protein" or "full-length receptor" refers to a receptor protein, such as a CD28 receptor protein. The term "full-length" encompasses receptor proteins lacking up to about 5 or up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus of the mature receptor protein after its signal peptide has been cleaved. For example, a specific cleavage site of the N-terminal signal peptide of a receptor can be defined, although variability in the exact point of cleavage has been observed. The term "full-length" does not refer to the presence or absence of amino acids in the N-terminal signal peptide of a receptor. In one embodiment, the term "full length" (e.g., a full-length CD28 or full-length CD40 intracellular domain according to certain aspects of the invention) encompasses a mature receptor protein (e.g., CD28 according to certain aspects of the invention) lacking up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein, after cleavage of that signal peptide. As noted above, a "full-length" CD28 receptor or other receptor or tumor antigen-binding domain according to various aspects of the invention does not include a signal peptide and may lack up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein (e.g., N-terminal residues N, K, I, L, and / or V). This is shown in exemplary fusions, e.g., SEQ ID NOs: 4-12 (note that these may lack up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein, as shown in the boxed region).

[0077] CoStAR has a modular format and can be constructed to include extracellular, transmembrane, and intracellular domains derived from one or more proteins, along with an scFv derived from an antibody that binds to a disease-associated antigen, e.g., a tumor-associated antigen. According to the present invention, in one embodiment, CoStAR comprises a disease-associated, e.g., tumor-associated antigen receptor, such as, but not limited to, a tumor-associated antigen-specific scFv, and a primary costimulatory receptor protein capable of binding to its cognate ligand and providing an intracellular signal. In some embodiments, the primary costimulatory receptor may be shorter than the full-length protein, but is sufficient to bind to and transmit a signal to its cognate ligand. In some embodiments, the primary costimulatory receptor domain is full-length, such as, but not limited to, full-length CD28. Thus, both the antigen-specific binding domain and the ligand-specific receptor can bind to their cognate antigen and ligand, respectively. The amino acid sequences provided herein provide several CoStAR construct embodiments. These include CoStAR constructs comprising an antigen-binding domain, an optional spacer, an optional costimulatory receptor protein comprising an extracellular ligand-binding segment or fragment thereof, and an intracellular CD40 signaling domain. In another embodiment, CoStAR comprises an antigen-binding domain, an optional spacer, an extracellular ligand-binding portion of a costimulatory receptor protein, a transmembrane domain, and an intracellular signaling domain of a selected costimulatory receptor protein, and an intracellular CD40 signaling domain. In some embodiments, the extracellular ligand-binding portion comprises a CD28 truncation, e.g., a C-terminal CD28 truncation after amino acid IEV, followed by the intracellular signaling domain. In some embodiments, the intracellular signaling domain is derived from CD40. The transmembrane domain separating the extracellular ligand-binding domain and the intracellular signaling domain may be derived exclusively from CD28 or CD40. In further embodiments, CoStAR may comprise an additional costimulatory domain, e.g., a third intracellular costimulatory signaling domain, and in this regard may resemble certain chimeric antigen receptors (CARs), which have been classified as first generation (CD3ζ only), second generation (one costimulatory domain + CD3ζ), or third generation (more than one costimulatory domain + CD3ζ).

[0078] Costimulatory receptor proteins useful in the CoStAR of the present invention include, but are not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6, which, in their native form, comprise an extracellular ligand-binding domain and an intracellular signaling domain. For example, CD2 is characterized as a cell adhesion molecule found on the surface of T cells and can initiate intracellular signals required for T cell activation. CD27 is characterized as a type II transmembrane glycoprotein belonging to the TNFR superfamily (TNFRSF), and its expression in B cells is induced by antigen receptor activation in B cells. CD28 is a protein on T cells and is a receptor for the CD80 (B7.1) and CD86 (B7.2) ligands on antigen-presenting cells. The CD137 (4-1BB) ligand is found on most leukocytes and some non-immune cells. The OX40 ligand is expressed on antigen-presenting cells, such as DC2 (dendritic cells), macrophages, and B lymphocytes. In one embodiment, the costimulatory receptor protein is full-length CD28 as defined herein.

[0079] CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily, and several isoforms are generated by alternative splicing. Its ligand, CD154 (also called CD40L), is a protein expressed primarily on activated T cells. For reference, the human CD40 isoform 1 protein sequence is set forth in GenBank accession number NP_001241.1 and includes a signal peptide (amino acids 1-20), a transmembrane domain (amino acids 194-215), and a cytoplasmic domain (amino acids 216-277) (SEQ ID NO: 32). CD40 receptor signaling involves adaptor proteins, including, but not limited to, TNF receptor-associated factors (TRAFs), and the CD40 cytoplasmic domain contains signaling components that contain amino acid sequences that match an SH3 motif (KPTNKAPH) (SEQ ID NO:35), a TRAF2 motif (PKQE (SEQ ID NO:36), PVQE (SEQ ID NO:37), SVQE (SEQ ID NO:38)), a TRAF6 motif (QEPQEINFP) (SEQ ID NO:39), and a PKA motif (KKPTNKA (SEQ ID NO:40), SRISVQE (SEQ ID NO:41)). The present invention further includes engineered signaling domains, e.g., engineered CD40 signaling domains, that contain TRAF-binding amino acid sequences. Engineered signaling domains that bind to TRAF1, TRAF2, TRAF3, and TRAF5 may comprise the major consensus sequence (P / S / A / T)X(Q / E)E or the minor consensus sequence PXQXXD, and may be identified in or derived from TNFR family members, such as, but not limited to, CD30, OX40, 41BB, and the EBV oncoprotein LMP1.(See, e.g., Ye, H et al., The Structural Basis for the Recognition of Diverse Receptor Sequences by TRAF2. Molecular Cell, 1999; 4(3):321-30. doi: 10.1016 / S1097-2765(00)80334-2; Park HH, Structure of TRAF Family: Current Understanding of Receptor Recognition. Front. Immunol. 2018; 9:1999. doi: 10.3389 / fimmu.2018.01999; Chung, JY et al., All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. Journal of Cell Science 2002; 115:679-688).

[0080] The examples disclosed herein demonstrate the operation of CD40 as a costimulatory signaling domain in CoStAR and further demonstrate that cytokine and chemokine expression profiles are altered by the selection of the signaling domain. In some embodiments, the costimulatory CD40 signaling domain of CoStAR promotes pro-inflammatory cytokines (e.g., IL-2, TNFα). In some embodiments, the costimulatory CD40 signaling domain of CoStAR reduces immunosuppressive cytokines (e.g., IL-5, IL-10). The costimulatory activity of a CD40 signaling domain or fragment may be observed in combination with a first receptor signaling domain such as, but not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6, compared to the activity of the first receptor signaling domain without the CD40 signaling domain or fragment. In this regard, the CD40 signaling domains of the present invention, which comprise signaling fragments containing specific factor-binding sites or in which specific factor-binding sites have been mutated, can, in combination with a costimulatory first signaling domain, enhance or suppress the relative expression of specific cytokines and / or chemokines compared to the activity of the first signaling domain of the costimulatory signaling domain alone.(See, for example, Ahonen, CL et al., The CD40-TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells. Nat Immunol. 2002; 3: 451-456, Mackey MF et al., Distinct contributions of different CD40 TRAF binding sites to CD154-induced dendritic cell maturation and IL-12 secretion. Eur J Immunol. 2003; 33: 779-789, Mukundan Let al., TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages. J Immunol. 2005; 174: 1081-1090).

[0081] In some embodiments, the CoStAR of the present invention comprises substantially all of the CD40 costimulatory domain. In some embodiments, the CoStAR of the present invention comprises two or more CD40 costimulatory domains. In some embodiments, the CoStAR of the present invention comprises a CD40 costimulatory domain signaling component or fragment or motif. In some embodiments, the CD40 signaling fragment or motif is an SH3-binding sequence (e.g., without limitation, KPTNKAPH (SEQ ID NO: 35), PTNKAPHP (SEQ ID NO: 443), or PTNKAPH (SEQ ID NO: 444)), a TRAF2 / TRAF3-binding sequence (e.g., without limitation, PKQE (SEQ ID NO: 506), PKQET (SEQ ID NO: 445), PVQE (SEQ ID NO: 507), PVQET (SEQ ID NO: 446), SVQE (SEQ ID NO: 508), SVQET (SEQ ID NO: 447)), a TRAF6-binding sequence (e.g., without limitation, PQEINF (SEQ ID NO: 509), QEPQEINF (SEQ ID NO: 448), or QEPQEINFP (SEQ ID NO: 39)), or a PKA sequence (e.g., without limitation, KKPTNKA (SEQ ID NO: 40), or SRISVQE (SEQ ID NO: 41), as well as two or more, or three or more, or four or fewer. or more such components or motifs, or combinations thereof, which may be in multiple copies and arranged in any order. In some embodiments, the CoStAR of the present invention comprises a CD40 costimulatory domain and a CD40 costimulatory domain signaling component or motif. In some embodiments, one or more of the SH3, TRAF2 / TRAF3, TRAF6, or PKA motifs of the CD40 signaling domain are mutated. In some embodiments, the SH3 motif, the TRAF2 / TRAF3 motif, and the TRAF6 motif are sufficient to modulate pro-inflammatory and / or immunosuppressive cytokines. In some embodiments, adding tandem copies of these motifs and / or mutating certain motifs amplifies these effects.

[0082] Table 1 provides non-limiting examples of TRAF2 / TRAF3 binding domains. Alignments of Table 1 with the minor and major consensus sequences are shown in Figures 82A-82B, respectively. [Table 1]

[0083] Thus, the TRAF2 / TRAF3 binding sequences of the present invention further include sequences such as P1V2Q3E4 and variants in which P1 is substituted with S, A, or T, V2 is substituted with Q, K, or E, Q3 is substituted with E, and / or E4 is substituted with A. In such variants, any one, two, three, or all four of P1V2Q3E4 may be substituted. Non-limiting examples are shown in Table 1 at positions P-2, P-1, P0, and P1. Illustrative, non-limiting examples of CD40 TRAF2 / TRAF3 sequence variants include the following, where the amino acids at P-2, P-1, P0, and P1 are boxed and the TRAF2 / TRAF3 source protein is identified:

[0084] [Table 2]

[0085] Table 3 provides non-limiting examples of TRAF6 binding sequences. An alignment of Table 3 with the major TRAF6 consensus sequence is shown in Figure 82C. [Table 3]

[0086] Illustrative, non-limiting examples of CD40 TRAF6 sequence variants include the following, with amino acids at P-2, P-1, P0, P1, P2, and P3 enclosed in dashed lines to identify the TRAF6 sequence origin: [Table 4]

[0087] [Table 5]

[0088] In some embodiments, the selection of one or more costimulatory domain signaling components or motifs is guided by the cells in which CoStAR is to be expressed and / or the desired costimulatory activity as more specifically specified by the signaling components or motifs, or the avoidance of costimulatory activity as more specifically specified by the signaling components or motifs. In some embodiments, the CoStAR signaling domain comprises a CD40 costimulatory domain or signaling component or motif thereof, or two or more such domains or components or motifs, or a combination thereof, as well as an additional full-length costimulatory domain or signaling component thereof derived from, without limitation, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6.

[0089] For reference, the human CD28 protein sequence is set forth in GenBank Accession No. NP_006130.1 and includes a signal peptide (amino acids 1-18), an extracellular domain (amino acids 19-152), a transmembrane domain (amino acids 153-179), and a cytoplasmic domain (amino acids 180-200). The extracellular domain contains an immunoglobulin-type domain (amino acids 21-136), including amino acids that constitute the antigen-binding site and the homodimer interface. The extracellular domain contains several asparagine residues that can be glycosylated, and the intracellular domain contains serine and tyrosine residues that can be phosphorylated. For reference, the human CD8 alpha chain protein sequence is set forth in GenBank Accession No. NP_001139345.1 and includes a signal peptide (amino acids 1-21), an extracellular domain (amino acids 22-182), a transmembrane domain (amino acids 183-203), and a cytoplasmic domain (amino acids 204-235). The extracellular domain contains an immunoglobulin-type domain (amino acids 28-128), including amino acids that constitute the antigen-binding site and the homodimer interface. The extracellular domain contains several asparagine residues that can be glycosylated, and the intracellular domain contains serine and tyrosine residues that can be phosphorylated.

[0090] For reference, the human IgG4 constant region sequence is described in UniProtKB / Swiss-Prot: Accession No. P01861.1 and includes CH1 (amino acids 1-98), hinge (amino acids 99-110), CH2 (amino acids 111-220), and CH3 (amino acids 221-327). The CH2 region contains an asparagine at amino acid 177, which is glycosylated and is associated with Fc receptors and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0091] For reference, the protein sequence of human CD137 (4-1BB), another TNFR superfamily member, is described under GenBank accession number NP_001552.2 and includes a signal peptide (amino acids 1-23), an extracellular domain (amino acids 24-186), a transmembrane domain (amino acids 187-213), and a cytoplasmic domain (amino acids 214-255). Binding of the CD137L ligand trimer expressed on antigen-presenting cells to CD137 results in receptor trimerization and activation of signaling cascades involved in T cell reactivity and survival (Li et al., "Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab." Cell Reports 2018;25:909-920). Co-immunoprecipitation of CD137 with the signaling adaptors TRAF-2 and TRAF-1 and the structural basis of the interaction have been reported (Ye, H et al., Molecular Cell, 1999; 4(3):321-30).

[0092] For reference, the human CD134 (OX40) protein sequence is described by GenBank accession number NP_003318.1 and includes a signal peptide (amino acids 1-28), an extracellular domain (amino acids 29-214), a transmembrane domain (amino acids 215-235), and a cytoplasmic domain (amino acids 236-277). This receptor has been shown to activate NF-kappaB through interaction with the adaptor proteins TRAF2 and TRAF5, and studies suggest that this receptor promotes the expression of the apoptosis inhibitors BCL2 and BCL2lL1 / BCL2-XL. The human T cell surface antigen CD2 has at least two isoforms. For reference, the human CD2 isoform 1 protein sequence is described by NP_001315538.1 and includes a signal peptide (amino acids 1-24), an extracellular domain (amino acids 25-235), a transmembrane domain (amino acids 236-261), and a cytoplasmic domain (amino acids 262-377). The human CD2 isoform 2 protein sequence is described by NP_001758.2.

[0093] For reference, the human CD357 (GITR) isoform-1 protein sequence is described by GenBank accession number NP_004186.1 and includes a signal peptide (amino acids 1-25), an extracellular domain (amino acids 26-162), a transmembrane domain (amino acids 163-183), and a cytoplasmic domain (amino acids 184-241). For reference, the human CD29 (beta 1 integrin) protein sequence is described by GenBank accession number NP_596867 and includes a signal peptide (amino acids 1-20), an extracellular domain (amino acids 21-728), a transmembrane domain (amino acids 729-751), and a cytoplasmic domain (amino acids 752-798).

[0094] The human CD150 (SLAM) protein sequence has several isoforms. In addition to the transmembrane form of CD150 (mCD150), hematopoietic cells express mRNA encoding a secreted form of CD150 (sCD150), which lacks the entire 30-amino acid transmembrane region. For reference, human SLAM isoform b is described by GenBank accession number NP_003028.1 and contains a signal peptide (amino acids 1-20), an extracellular domain (amino acids 21-237), a transmembrane domain (amino acids 238-258), and a cytoplasmic domain (amino acids 259-335). Human SLAM isoforms are described by GenBank accession number NP_001317683.1.

[0095] CD278 or ICOS (Inducible T cell COStimulator) is a costimulatory molecule of the CD28 superfamily expressed on activated T cells. The human ICOS precursor (199 amino acids with a signal peptide) is described by GenBank accession number NP_036224.1 and contains a signal peptide (amino acids 1-20), an Ig-V-like domain (amino acids 21-140), a transmembrane domain (amino acids 141-161), and an intracellular domain (amino acids 162-199). ICOS contains the IProx motif sequence SSSVHDPNGE (SEQ ID NO: 466). The IProx motif sequence SSSXXXPXGE (SEQ ID NO: 467) resembles certain binding sites in TRAF1 (SASFQRPQSE (SEQ ID NO: 468)), TRAF2 (SSSFQRPVND (SEQ ID NO: 469)), TRAF3 (SSFKKPTGE (SEQ ID NO: 470)), and TRAF5 (SSSFKRPDGE (SEQ ID NO: 471)).

[0096] Hematopoietic cell signaling factor (HCST), also known as DAP10, KAP10, PIK3AP, and hematopoietic cell signaling factor (GenBank accession number NP_055081.1), encodes a transmembrane signaling adaptor that is thought to form part of a receptor complex with the C-type lectin-like receptor NKG2D. The intracellular domain contains a YxxM motif, a phosphatidylinositol 3-kinase binding site.

[0097] In an embodiment of the present invention, CoStAR can be expressed alone under the control of a promoter in a therapeutic cell population, such as tumor-infiltrating lymphocytes (TILs), having therapeutic activity. Alternatively, CoStAR can be expressed together with a therapeutic transgene, such as a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). (Note that up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids may be missing from the N-terminus of the mature receptor protein.) Thus, in one aspect, the present invention also relates to CoStAR constructs having a sequence set forth in any of SEQ ID NOS: 42-185, 192-335, and 344-430, including sequences missing up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids from the N-terminus of the mature receptor protein. Suitable TCRs and CARs, such as HLA-A*02-NYESO-1-specific TCRs (Rapoport et al. Nat Med 2015) or anti-CD19scFv.CD3ζ fusion CARs (Kochenderfer et al. J Clin Oncol 2015), are well known in the literature and have been successfully used to treat myeloma or B-cell malignancies, respectively. The CoStAR described herein can be expressed with any known CAR or TCR, thus providing cells with a tunable proliferation switch to enable cell expansion in vitro or in vivo, and a conventional activation mechanism in the form of a TCR or CAR for anti-cancer activity. Thus, the present invention provides cells for use in adoptive cell therapy comprising the CoStAR described herein and a TCR and / or CAR that specifically binds to a tumor-associated antigen. Exemplary CoStARs, including CD28, include an extracellular antigen-binding domain and extracellular, transmembrane, and intracellular signaling domains.

[0098] The term "antigen-binding domain," as used herein, refers to an antibody fragment, including, but not limited to, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv), bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not constitute a complete antibody structure. The antigen-binding domain can bind to the same antigen as that bound by the parent antibody or parent antibody fragment (e.g., the parent scFv). In some embodiments, the antigen-binding fragment can comprise one or more complementarity-determining regions (CDRs) from a particular human antibody grafted onto frameworks (FRs) from one or more different human antibodies. The "antigen-binding domain" may also be referred to as a "ligand-binding domain."

[0099] The antigen-binding domain can be made specific for any disease-associated antigen, including, but not limited to, tumor-associated antigens (TAA) and infectious disease-associated antigens. In some embodiments, the ligand-binding domain is bispecific. Antigens have been identified in most human cancers, including Burkitt's lymphoma, neuroblastoma, melanoma, osteosarcoma, renal cell carcinoma, breast cancer, prostate cancer, lung cancer, and colon cancer. TAAs include, but are not limited to, CD19, CD20, CD22, CD24, CD33, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2. TAAs further include neoantigens, peptide / MHC complexes, and HSP / peptide complexes.

[0100] In some embodiments, the antigen-binding domain comprises a T cell receptor or binding fragment thereof that binds to a defined tumor-specific peptide-MHC complex. The term "T cell receptor" or "TCR" refers to a heterodimeric receptor composed of paired αβ or γδ chains on the surface of a T cell. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through complementarity-determining regions (CDRs), followed by a constant domain (C) that is anchored to the cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunit of the CD3 signaling apparatus. Each V domain has three CDRs. These CDRs interact with a complex of antigenic peptides bound to proteins encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, 868 p.).

[0101] In some embodiments, the antigen-binding domain comprises a natural ligand of a protein expressed in tumors or a tumor-binding fragment thereof. A non-limiting example is PD1, which binds to PDL1. Another example is transferrin receptor 1 (TfR1), also known as CD71, a homodimeric protein that is a key regulator of intracellular iron homeostasis and proliferation. TfR1 is expressed at low levels in a wide range of cells, but at high levels in rapidly proliferating cells, including malignant tumor cells, where overexpression is associated with poor prognosis. In some embodiments of the present invention, the antigen-binding domain comprises transferrin or a transferrin receptor-binding fragment thereof.

[0102] In some embodiments, the antigen-binding domain is specific for a defined tumor-associated antigen, such as, but not limited to, FRα, CEA, 5T4, CA125, SM5-1, or CD71. In some embodiments, the tumor-associated antigen can be a tumor-specific peptide-MHC complex. In certain such embodiments, the peptide is a neoantigen. In other embodiments, the tumor-associated antigen is a peptide-heat shock protein complex. In various embodiments, the present invention provides a CoStAR comprising: i. An scFv that binds to carcinoembryonic antigen (CEA), the spacer and transmembrane sequences of CD28, the CD28 signaling domain, and the CD40 signaling domain. ii. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, and the CD40 signaling domain. iii. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, the CD137 signaling domain, and the CD40 signaling domain.

[0103] iv. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, the CD134 signaling domain, and the CD40 signaling domain. v. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, the CD2 signaling domain, and the CD40 signaling domain. vi. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, the GITR signaling domain, and the CD40 signaling domain. vii. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, the CD29 signaling domain, and the CD40 signaling domain.

[0104] viii. An scFv that binds to CEA, the spacer and transmembrane sequences of CD28, the CD150 signaling domain, and the CD40 signaling domain. ix. An scFv that binds CEA, the spacer and transmembrane sequences of CD8, the CD28 signaling domain, and the CD40 signaling domain. x. An scFv that binds to CEA, the spacer and transmembrane sequences of CD8, and the CD40 signaling domain. xi. An scFv that binds to CEA, the spacer and transmembrane sequences of CD8, the CD137 signaling domain, and the CD40 signaling domain.

[0105] xii. An scFv that binds to CEA, the spacer and transmembrane sequences of CD8, the CD134 signaling domain, and the CD40 signaling domain. xiii. An scFv that binds to CEA, the spacer and transmembrane sequences of CD8, the CD2 signaling domain, and the CD40 signaling domain. xiv. scFv that binds to CEA, the spacer and transmembrane sequences of CD8, the GITR signaling domain, and the CD40 signaling domain. xv. scFv that binds to CEA, the spacer and transmembrane sequences of CD8, the CD29 signaling domain, and the CD40 signaling domain.

[0106] xvi. An scFv that binds to CEA, the spacer and transmembrane sequences of CD8, the CD150 signaling domain, and the CD40 signaling domain. xvii. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD28 signaling domain, and a CD40 signaling domain. xviii. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, and a CD40 signaling domain. xix. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD137 signaling domain, and a CD40 signaling domain.

[0107] xx. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD134 signaling domain, and a CD40 signaling domain. xxi. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD2 signaling domain, and a CD40 signaling domain. xxii. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a GITR signaling domain, and a CD40 signaling domain. xxiii. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD29 signaling domain, and a CD40 signaling domain. xxiv. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD150 signaling domain, and a CD40 signaling domain.

[0108] xxv. An scFv that binds CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a first CD40 signaling domain, and a second CD40 signaling domain. xxvi. An scFv that binds to CEA, a spacer comprising an IgG4 constant region and a CD28 transmembrane sequence, a first CD40 signaling domain, and a second variant CD40 signaling domain. xxvii. A binding domain that binds to PDL1, a short spacer and transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain. xxviii. A binding domain that binds to PDL1, a short spacer and transmembrane sequence of CD28, and a CD40 signaling domain.

[0109] xxix. A binding domain that binds to CD155, CD112, or CD113, a CD28 transmembrane domain, a CD28 signaling domain, and a CD40 signaling domain. xxx. A binding domain that binds to CD155, CD112, or CD113, a CD28 transmembrane domain, and a CD40 signaling domain. xxxi. An scFv that binds to CEA, a binding domain that binds to PDL1, a short spacer and transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain. xxxii. An scFv that binds to CEA, a binding domain that binds to PDL1, a short spacer and transmembrane sequence of CD28, and a CD40 signaling domain.

[0110] xxxiii. An scFv that binds to CEA, a binding domain that binds to CD155, CD112, or CD113, a short spacer and transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain. xxxiv. An scFv that binds to CEA, a binding domain that binds to CD155, CD112, or CD113, a short spacer and transmembrane sequence of CD28, and a CD40 signaling domain. xxxv. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, and an NTRK1 signaling domain. xxxvi. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, an NTRK1 signaling domain, and a CD40 signaling domain.

[0111] xxxvii. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, and an NTRK1 signaling domain. xxxviii. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, an NTRK1 signaling domain, and a CD40 signaling domain. xxxix. An scFv that binds to CEA, a spacer comprising the ICOS extracellular domain and transmembrane sequence of ICOS, an ICOS signaling domain, and a CD40 signaling domain. xl. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, and an ICOS signaling domain.

[0112] xli. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, an ICOS signaling domain, and a CD40 signaling domain. xlii. An scFv that binds to CEA, a spacer comprising the CD2 extracellular domain and transmembrane sequence of CD2, a CD2 signaling domain, and a CD40 signaling domain. xliii. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, and a CD2 signaling domain. xliv. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, a CD40 signaling domain, and a CD2 signaling domain.

[0113] xlv. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, and a CD137 signaling domain. xlvi. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, a CD40 signaling domain, and a CD137 signaling domain. xlvii. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, and a DAP10 signaling domain. xlviii. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, a CD40 signaling domain, and a DAP10 signaling domain.

[0114] xlix. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD28 signaling domain, and a CD134 signaling domain. xlx. An scFv that binds to CEA, a spacer comprising the CD28 extracellular domain and transmembrane sequence of CD28, a CD40 signaling domain, and a CD134 signaling domain. In some embodiments, the present invention provides a CoStAR comprising an scFv that binds to MSLN linked to the spacer, transmembrane, and signaling domain structure of any one of paragraphs i to xlx. In some embodiments, the present invention provides a CoStAR comprising an scFv that binds to FolR1 linked to the spacer, transmembrane, and signaling domain structures of any one of paragraphs i to xlx.

[0115] In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i to xxxiv, wherein the CoStAR binds to FolR1 via a binding domain comprising an antigen-binding fragment of scFv MOV19 (SEQ ID NO: 9). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures of i-xxxiv, wherein the CoStAR binds to CEA via a binding domain comprising an antigen-binding fragment of scFv MFE23 (SEQ ID NO: 10). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to CEA via a binding domain comprising an antigen-binding fragment of scFv MFE23(K>Q) (SEQ ID NO: 11).

[0116] In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to CEA via a binding domain comprising an antigen-binding fragment of humanized scFv MFE23 (hMFE23) (SEQ ID NO: 12). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to CEA via a binding domain comprising an antigen-binding fragment of CEA6 (SEQ ID NO: 13). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to CEA via a binding domain comprising an antigen-binding fragment of scFv BW431 / 26 (SEQ ID NO: 14).

[0117] In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to CEA via a binding domain comprising an antigen-binding fragment of scFv HuT84.66(M5A) (SEQ ID NO: 15). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i to xxxiv, wherein the CoStAR binds to FolR1 via a binding domain comprising an antigen-binding fragment of scFv MOV19 (SEQ ID NO: 9). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures of i-xxxiv and xxxi-xxxiv, wherein the CoStAR binds to MSLN via a binding domain comprising an antigen-binding fragment of SS1 (SEQ ID NO: 186).

[0118] In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to MSLN via a binding domain comprising an antigen-binding fragment of scFv M5 (humanized SS1) (SEQ ID NO: 187). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to MSLN via a binding domain comprising an antigen-binding fragment of humanized scFv HN1 (SEQ ID NO: 188). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to MSLN via a binding domain comprising an antigen-binding fragment of M912 (SEQ ID NO: 189).

[0119] In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to MSLN via a binding domain comprising an antigen-binding fragment of HuYP218 (SEQ ID NO: 190). In some embodiments, the present invention provides a CoStAR comprising any one of the spacer, transmembrane, and signaling domain structures i-xxvi and xxxi-xxxiv, wherein the CoStAR binds to MSLN via a binding domain comprising an antigen-binding fragment of scFv P4 (SEQ ID NO: 191).

[0120] In some embodiments, any of the above (including I-xlx) can be used with a CD40, TRAF2, TRAF2 / 3, or TRAF6 variant provided herein, including those in Figures 28, 67, 77A-77C, 83-89D, and 92 (except those in Figures 78 and 79), and in Tables 2, 4-5, and 7-14, and SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences A-C.

[0121] As used herein, the terms "specifically binds to" or "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody or antibody portion, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody portion that specifically binds to a target (which may be an epitope) is an antibody portion that binds to the target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, an antibody portion that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (e.g., a cell surface antigen or a peptide / MHC protein complex) with a binding affinity that is at least about 10 times its binding affinity for other targets.

[0122] Spacer The CoStAR of the present invention may comprise a spacer region between the antigen-binding domain and the costimulatory receptor. As used herein, the term "spacer" refers to the extracellular structural region of CoStAR that separates the antigen-binding domain from the external ligand-binding domain of the costimulatory protein. The spacer provides flexibility for accessing the targeted antigen and receptor ligand. In some embodiments, a long spacer is used to target, for example, membrane-proximal epitopes or glycosylated antigens (see Guest RD et al. The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J. Immunother. 2005;28:203-211; Wilkie S. et al., Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor. J. Immunol. 2008;180:4901-4909).In other embodiments, CoStAR has a short spacer, e.g., to target a membrane-distal epitope (see Hudecek M. et al., Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013;19:3153-3164; Hudecek M. et al., The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol. Res. 2015;3:125-135). In some embodiments, the spacer comprises or is derived from all or part of an IgG hinge, including, but not limited to, IgG1, IgG2, or IgG4. "Derived from an Ig hinge" means that the spacer comprises an insertion, deletion, or mutation in the IgG hinge. In some embodiments, the spacer may comprise all or a portion of one or more antibody constant domains, such as, but not limited to, a CH2 and / or CH3 domain. In some embodiments, in spacers comprising all or a portion of a CH2 domain, the CH2 domain is modified so that it does not bind to Fc receptors. For example, Fc receptor binding in myeloid cells has been found to impair CAR T cell functionality. In some embodiments, the spacer comprises all or a portion of an Ig-like hinge derived from CD28, CD8, or other proteins containing a hinge region. In some embodiments of the invention comprising a spacer, the spacer is 1 to 50 amino acids in length.

[0123] In non-limiting embodiments, the spacer comprises essentially all of an extracellular domain, e.g., the CD28 extracellular domain (i.e., from about amino acid 19, 20, 21, or 22 to about amino acid 152), or the extracellular domain of another protein, including, but not limited to, another TNFR superfamily member. In some embodiments, the spacer comprises a portion of the extracellular domain, e.g., a portion of the CD28 extracellular domain, optionally lacking all or most of the Ig domain. In another embodiment, the spacer comprises amino acids from about 141 to about 152 of CD28, but not other portions of the CD28 extracellular domain. In another embodiment, the spacer comprises amino acids from about 128 to about 182 of CD8, but not other portions of the CD8 extracellular domain. Linker

[0124] In some embodiments, the CoStAR extracellular domain comprises a linker. A linker comprises a short segment of amino acids used to connect a domain, e.g., a binding domain, to a spacer or transmembrane domain. To allow flexibility for ligand binding, the ligand-binding domain is typically connected to the spacer or transmembrane domain by a flexible linker comprising approximately 5-25 amino acids, e.g., AAAGSGGSG (SEQ ID NO: 18), GGGGSGGGSGGGGS (SEQ ID NO: 431), etc. In some embodiments, the CoStAR comprises a binding domain directly connected to the transmembrane domain by a linker, without a spacer. In some embodiments, the CoStAR comprises a binding domain directly connected to the transmembrane domain by a spacer, without a linker.

[0125] Signaling domains As described above, in some embodiments, CoStAR comprises a full-length primary costimulatory receptor, which may include, without limitation, an extracellular ligand-binding portion and an intracellular signaling portion of CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. In other embodiments, the costimulatory receptor comprises a chimeric protein, for example, comprising the extracellular ligand-binding domain of one of the aforementioned proteins and the intracellular signaling domain of another of the aforementioned proteins. In some embodiments, the signaling portion of CoStAR comprises a single signaling domain. In other embodiments, the signaling portion of CoStAR comprises a second intracellular signaling domain, such as, but not limited to, CD2, CD27, CD28, CD40, CD134 (OX40), CD137 (4-1BB), or CD150 (SLAM). In some embodiments, the first and second intracellular signaling domains are the same. In other embodiments, the first and second intracellular signaling domains are different. In some embodiments, the costimulatory receptor is capable of dimerization. Without being bound by theory, it is believed that CoStAR dimerizes or associates with other auxiliary molecules to initiate a signal. In some embodiments, CoStAR dimerizes or associates with auxiliary molecules through transmembrane domain interactions. In some embodiments, dimerization or association with auxiliary molecules is assisted by costimulatory receptor interactions at the intracellular and / or extracellular portions of the costimulatory receptor.

[0126] Transmembrane domain Although the primary function of the transmembrane is to anchor CoStAR to the T cell membrane, in some embodiments, the transmembrane domain influences CoStAR function. In some embodiments, the transmembrane domain is comprised by the full-length primary costimulatory receptor domain. In embodiments of the present invention in which a CoStAR construct comprises the extracellular domain of one receptor and the intracellular signaling domain of a second receptor, the transmembrane domain may be that of the extracellular domain or that of the intracellular domain. In some embodiments, the transmembrane domain is derived from CD4, CD8α, CD28, or ICOS. Gueden et al. have linked the use of the ICOS transmembrane domain to increased CAR T cell persistence and overall anti-tumor efficacy (Guedan S. et al., Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight. 2018;3:96976). In some embodiments, the transmembrane domain comprises a hydrophobic α-helix that spans the cell membrane.

[0127] In some embodiments, the transmembrane domain comprises amino acids from about amino acid 153 to about amino acid 179 of the CD28 transmembrane domain. In another embodiment, the transmembrane domain comprises amino acids from about amino acid 183 to about amino acid 203 of the CD8 transmembrane domain. In some embodiments, the CoStAR of the invention may comprise several amino acids between the transmembrane domain and the signaling domain. For example, in one construct described herein, the link from the CD8 transmembrane domain to the signaling domain comprises several amino acids of the CD8 cytoplasmic domain (e.g., amino acids 204-210 of CD8).

[0128] variant In some embodiments, amino acid sequence variants of the antibody moieties or other moieties provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody moiety. Amino acid sequence variants of the antibody moiety can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody moiety, or by peptide synthesis. Such modifications include, for example, deletion of, and / or insertion of, and / or substitution of, residues within the amino acid sequence of the antibody moiety. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0129] In some embodiments, antibody-binding domain portions containing one or more amino acid substitutions, deletions, or insertions are provided. Targeted sites for mutational changes include the heavy and light chain variable regions (VRs) and framework regions (FRs) of the antibody-binding domain. Amino acid substitutions can be introduced into the binding domain of interest, and the products screened for desired activity, such as retained / improved antigen binding or reduced immunogenicity. In some embodiments, amino acid substitutions can be introduced into one or more of the primary costimulatory receptor domain (extracellular or intracellular), the secondary costimulatory receptor domain, or the extracellular co-receptor domain. Thus, the present invention encompasses the CoStAR proteins and component parts specifically disclosed herein, as well as variants thereof, i.e., CoStAR proteins and component parts having at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences specifically disclosed herein. The terms "percent similarity," "percent identity," and "percent homology," when referring to specific sequences, are used as described in the University of Wisconsin GCG software program BestFit. Other algorithms may also be used, such as BLAST, psiBLAST, or TBLASTN (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448).

[0130] A particular amino acid sequence variant may differ from a reference sequence by the insertion, addition, substitution, or deletion of one amino acid, two, three, four, five to ten, ten to twenty, or twenty to thirty amino acids. In some embodiments, a variant sequence may comprise a reference sequence in which one, two, three, four, five, six, seven, eight, nine, ten, or more residues have been inserted, deleted, or substituted. For example, five, ten, fifteen, up to twenty, up to thirty, or up to forty residues may be inserted, deleted, or substituted.

[0131] In some preferred embodiments, a variant may differ from a reference sequence by one, two, three, four, five, six, seven, eight, nine, ten, or more conservative substitutions. Conservative substitutions involve the replacement of one amino acid with a different amino acid with similar properties. For example, an aliphatic residue may be replaced by another aliphatic residue, a non-polar residue may be replaced by another non-polar residue, an acidic residue may be replaced by another acidic residue, a basic residue may be replaced by another basic residue, a polar residue may be replaced by another polar residue, or an aromatic residue may be replaced by another aromatic residue. Conservative substitutions may be, for example, between amino acids within the following groups:

[0132] Conservative substitutions are shown in Table 6 below. [Table 6]

[0133] Amino acids can be divided into different classes according to common side chain properties: a. hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, c. acidic: Asp, Glu, d. basic: His, Lys, Arg, e. residues that affect chain orientation: Gly, Pro, aromatic: Trp, Tyr, Phe. Non-conservative substitutions would involve exchanging any member of one of these classes for another class. cell The cells used in the present invention can be any lymphocyte useful in adoptive cell therapy, such as T cells or natural killer (NK) cells, NKT cells, gamma / delta T cells, or regulatory T cells. The cells can be allogeneic or autologous to the patient.

[0134] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on the cell surface. There are various types of T cells, as summarized below. Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in graft rejection. CTLs express CD8 molecules on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0135] Memory T cells are a subset of antigen-specific T cells that persist long after an infection has cleared. They rapidly expand to large numbers of effector T cells upon re-exposure to cognate antigen, providing the immune system with a "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO. Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity toward the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0136] Two major classes of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells. + CD25 + FoxP3 + Treg cells (also known as Treg cells) originate in the thymus and interact with developing T cells and TSLP-activated myeloid dendritic cells (CD11c + ) and plasma cells, dendritic cells (CD123 + ) and the immune system. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Adaptive Treg cells (also known as Tr1 or Th3 cells) can arise during a normal immune response. Natural killer cells (or NK cells) are a type of cytolytic cell that forms part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner. NK cells (belonging to the class of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute the third type of cell that differentiates from common lymphoid progenitor cells that give rise to B and T lymphocytes.

[0137] In some embodiments, the therapeutic cells of the present invention comprise autologous cells engineered to express CoStAR. In some embodiments, the therapeutic cells of the present invention comprise allogeneic cells engineered to express CoStAR. Autologous cells expressing CoStAR may be advantageous in avoiding graft-versus-host disease (GVHD) due to TCR-mediated recognition of recipient alloantigens. In addition, the immune system of the CoStAR recipient may attack the injected CoStAR cells, causing rejection. In certain embodiments, to prevent GVHD and reduce rejection, endogenous TcRs are removed from allogeneic CoStAR cells by genome editing.

[0138] nucleic acid Aspects of the present invention provide nucleic acid sequences of the present invention that encode any of the CoStAR, polypeptides, or proteins (including functional portions and functional variants thereof) described herein. As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other. Those skilled in the art will understand that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, those skilled in the art will understand that nucleotide substitutions can be made using routine techniques to reflect the codon usage of any particular host organism in which the polypeptide will be expressed, e.g., codon optimization, without affecting the polypeptide sequence encoded by the polynucleotides described herein. Numerous different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of the polynucleotide of interest.

[0139] The terms "variant," "homologue," or "derivative" in reference to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one or more nucleic acid residues from or to that sequence. The nucleic acid sequence can encode a CoStAR protein including, without limitation, any one of SEQ ID NOs: 42-247 or a variant thereof. The nucleotide sequence can include the indicated codon-optimized nucleic acid sequence engineered for expression in human cells. The present invention also provides nucleic acid sequences, including those encoding CoStAR, and additional nucleic acid sequences encoding a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0140] The nucleic acid sequences may be linked by a sequence that allows for co-expression of two or more nucleic acid sequences. For example, the construct may include an internal promoter, an internal ribosome entry sequence (IRES) sequence, or a sequence encoding a cleavage site. The cleavage site may be self-cleaving, such that once the polypeptide is produced, it is immediately cleaved into separate proteins without the need for any external cleavage activity. Various self-cleaving sites are known, including the foot-and-mouth disease virus (FMDV) and 2A self-cleaving peptides. The co-expression sequence may be an internal ribosome entry sequence (IRES). The co-expression sequence may be an internal promoter.

[0141] vector In one aspect, the present invention provides a vector comprising a nucleic acid sequence or nucleic acid construct of the present invention. Such vectors can be used to introduce nucleic acid sequences or nucleic acid constructs into host cells so that the host cells express one or more CoStARs according to the first aspect of the invention, and optionally one or more other proteins of interest (POIs), such as TCRs or CARs. The vectors can be, for example, plasmids or viral vectors, such as retroviral or lentiviral vectors, or transposon-based vectors or synthetic mRNAs. The nucleic acids of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0142] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of one or more transgenes and the propagation of the transgenes in daughter cells. The vectors may be capable of transfecting or transducing lymphocytes, including T cells or NK cells. The present invention also provides vectors into which the nucleic acids of the present invention are inserted. Expression of natural or synthetic nucleic acids encoding CoStAR and optionally TCR or CAR is typically achieved by operably linking nucleic acids encoding CoStAR and TCR / CAR polypeptides or portions thereof to one or more promoters and incorporating the construct into an expression vector. Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. In many cases, the spacing between promoter elements is flexible, so that promoter function is maintained even when elements are inverted or moved relative to one another. In the case of the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0143] One example of a suitable promoter is the cytomegalovirus (CMV) immediate-early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, MSCV promoter, MND promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0144] The vector may be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals; see also WO01 / 96584, WO01 / 29058, and U.S. Pat. No. 6,326,193. In some embodiments, the expressed construct is set forth in SEQ ID NOs: 42-247. In some embodiments, the nucleic acid is a multicistronic construct, allowing expression of multiple transgenes (e.g., CoStAR and TCR and / or CAR) under the control of a single promoter. In some embodiments, the transgenes (e.g., CoStAR and TCR and / or CAR) are separated by a self-cleaving 2A peptide. Examples of 2A peptides useful in the nucleic acid constructs of the present invention include F2A, P2A, T2A, and E2A. In another embodiment of the present invention, the nucleic acid construct of the present invention is a multicistronic construct containing two promoters, one promoter driving CoStAR expression and the other promoter driving TCR or CAR expression. In some embodiments, the dual promoter construct of the present invention is unidirectional. In other embodiments, the dual promoter construct of the present invention is bidirectional. For the purpose of evaluating the expression of a CoStAR polypeptide or a portion thereof, the expression vector introduced into cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population to be transfected or transduced via a viral vector.

[0145] Cell Source Prior to expansion and genetic modification, a source of cells (e.g., immune effector cells, e.g., T cells or NK cells) is obtained from a subject. The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL™ gradient centrifugation or counterflow centrifugal elution. T cells can be collected at apheresis centers and cell storage facilities where T cells can be collected, maintained, and easily transported. T cells can be cryopreserved and stored for later use. Acceptable storage periods can be determined and verified, and may be up to six months, up to one year, or longer.

[0146] In some embodiments, tumor-infiltrating cells (TILs) are isolated and / or expanded from tumors, for example, by fragmenting, sectioning, or enzymatically digesting tumor biopsies or tumor masses. TILs can be produced in a two-stage process using tumor biopsies as starting material: Stage 1 (typically taking 2-3 hours) involves the initial collection and processing of tumor material using sectioning, enzymatic digestion, and homogenization to obtain a single-cell suspension that can be directly cryopreserved to stabilize the starting material for subsequent manufacturing; Stage 2 can occur days or years later. Stage 2 can occur over a 4-week period, which can be a continuous process beginning with thawing the Stage 1 product and expanding TILs from the tumor starting material (approximately 2 weeks), followed by a rapid expansion process of TIL cells (approximately 2 weeks) to increase the quantity of cells and thus the dose. TILs can be concentrated and washed before being formulated as a liquid cell suspension.

[0147] TIL populations can be transduced at any time after collection. In some embodiments, cryopreserved TIL populations are transduced to express CoStAR after thawing. In some embodiments, TIL populations are transduced to express CoStAR during growth or initial expansion from tumor starting material. In some embodiments, TIL populations are transduced to express CoStAR during REP, for example, but not limited to, about day 8 to about day 10 of REP. Exemplary TIL preparations are described in applicant's U.S. patent application Ser. No. 62 / 951,559, filed Dec. 20, 2019.

[0148] Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubating with anti-CD3 / anti-CD28 conjugated beads, e.g., DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. In one embodiment, this period is approximately 30 minutes. In a further embodiment, this period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In a further embodiment, this period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another preferred embodiment, this period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. In any situation where T cells are scarce compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals, longer incubation times may be used to isolate T cells. Furthermore, using longer incubation times may increase the efficiency of CD8+ T cell capture. Thus, simply by shortening or lengthening the time T cells are bound to CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially selected for or against either at the initiation of culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or against either at the initiation of culture or at other desired times. Those skilled in the art will understand that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells during the activation and expansion process. Alternatively, the "unselected" cells may be subjected to additional rounds of selection.

[0149] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers specific to the cells being negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25-conjugated beads or other similar selection methods.

[0150] The methods described herein can include selecting a particular subpopulation of immune effector cells, e.g., T cells (a regulatory T cell-depleted population, a CD25+ depleted cell), e.g., using, e.g., negative selection techniques, e.g., those described herein. Preferably, the population of regulatory T cell-depleted cells comprises less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells.

[0151] Specific subpopulations of CoStAR effector cells that specifically bind to a target antigen can be enriched by positive selection techniques. For example, in some embodiments, effector cells are enriched by incubating with target antigen-conjugated beads for a period sufficient for positive selection of the desired abTCR effector cells. In some embodiments, this period is approximately 30 minutes. In some embodiments, this period ranges from 30 minutes to 36 hours or more (including all ranges between these values). In some embodiments, this period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In some embodiments, this period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For the isolation of effector cells present at low levels in a heterogeneous cell population, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate effector cells in any situation where effector cells are present at low levels compared to other cell types. Those skilled in the art will appreciate that multiple rounds of selection can also be used in the context of the present invention.

[0152] Alternatively, T cells for stimulation can be frozen after a washing step. After removing plasma and platelets by a washing step, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may also be used, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen.

[0153] Allogeneic CoStAR In embodiments described herein, the immune effector cells can be allogeneic immune effector cells, e.g., T cells or NK cells. For example, the cells can be allogeneic T cells, e.g., allogeneic T cells that lack expression of an endogenous T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II. A T cell that lacks a functional endogenous TCR can, for example, be engineered not to express any functional TCR on its surface, engineered not to express one or more subunits that comprise a functional TCR (e.g., engineered not to express (or exhibit reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and / or TCR zeta), or engineered to produce little to no functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, for example, by expression of a mutated or truncated form of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that the TCR does not elicit a deleterious immune response in the host.

[0154] The T cells described herein can be engineered, for example, to not express functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate cell surface expression of HLA, e.g., HLA class 1 and / or HLA class II. In some embodiments, downregulation of HLA can be achieved by reducing or eliminating expression of beta 2 microglobulin (B2M).

[0155] In some embodiments, T cells can lack a functional TCR and a functional HLA, e.g., HLA class I and / or HLA class II. Engineered T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0156] In some embodiments, the allogeneic cells may be cells that do not express or express at low levels of inhibitory molecules, such as cells engineered by any of the methods described herein. For example, the cells may be cells that do not express or express at low levels of inhibitory molecules that can reduce the ability of CoStAR-expressing cells to initiate immune effector responses. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta. Inhibition of inhibitory molecules, for example, by inhibition at the DNA, RNA, or protein level, can optimize the performance of CAR-expressing cells. In embodiments, inhibitory nucleic acids can be used, e.g., inhibitory nucleic acids such as dsRNA, e.g., siRNA or shRNA, as described herein by way of example, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs).

[0157] Use of siRNA or shRNA to inhibit endogenous TCR or HLA In some embodiments, TCR expression and / or HLA expression can be inhibited in T cells using siRNA or shRNA targeting nucleic acids encoding TCR and / or HLA, and / or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta). Expression of siRNA and shRNA in T cells can be achieved using any conventional expression system, such as a lentiviral expression system. Exemplary shRNAs that downregulate the expression of TCR components are described, for example, in U.S. Publication No. 2012 / 0321667. Exemplary siRNA and shRNAs that downregulate the expression of HLA class I and / or HLA class II genes are described, for example, in U.S. Publication No. 2007 / 0036773. CRISPR that inhibits TCR or HLA

[0158] As used herein, "CRISPR" or "TCR and / or HLA-inhibiting CRISPR" refers to a set of clustered regularly interspaced short palindromic repeats or a system containing such a set of repeats. "Cas," as used herein, refers to a CRISPR-associated protein. "CRISPR / Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate TCR and / or HLA genes and / or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta).

[0159] Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune cell system that confers resistance to foreign genetic elements such as plasmids and phages, providing a form of adaptive immunity. Barrangou et al. (2007) Science 315: 1709-1712, Marragini et al. (2008) Science 322: 1843-1845. T cell activation and expansion T cells can generally be activated and expanded using methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0160] Generally, T cells of the present invention can be expanded by contacting the T cell surface with a surface bound to an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules. Specifically, a T cell population can be stimulated, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, as described herein, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. Anti-CD3 and anti-CD28 antibodies can be used to stimulate proliferation of either CD4+ or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and can be used, as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0161] In some embodiments, expansion can be performed using flasks or vessels or gas permeable vessels known to those of skill in the art and can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, or about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0162] In some embodiments, expansion can be performed using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15), such as an anti-CD3 antibody, e.g., about 30 ng / ml OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, Calif.), or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA). CoStAR cells can be expanded in vitro by including one or more cancer antigens, e.g., antigenic portions thereof, e.g., epitopes, expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35 (27L) or gp100:209-217 (210M), optionally in the presence of a T cell growth factor, e.g., 300 IU / mL IL-2 or IL-15. Other suitable antigens include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. CoStAR cells can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, CoStAR cells may be further stimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, stimulation occurs as part of expansion. In some embodiments, expansion occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0163] In some embodiments, the cell culture medium comprises IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7 Contains 500 IU / mL, or about 8000 IU / mL, or between 1000-2000 IU / mL, between 2000-3000 IU / mL, between 3000-4000 IU / mL, between 4000-5000 IU / mL, between 5000-6000 IU / mL, between 6000-7000 IU / mL, between 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0164] In some embodiments, the cell culture medium comprises an OKT3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 120 ng / mL, about 140 ng / mL, about 160 ng / mL, about 180 ng / mL, about 190 ng / mL, about 210 ng / mL, about 220 ng / mL, about 230 ng / mL, about 240 ng / mL, about 250 ng / mL, about 260 ng / mL, about 270 ng / mL, about 280 ng / mL, about 290 ng / mL, about 310 ng / mL, about 320 ng / mL, about 330 ng / mL, about 340 ng / mL, about 350 ng / mL, about 360 ng / mL, about 370 ng / mL, about 380 ng / mL, about 390 ng / mL, about 400 ng / mL, about 420 ng / mL, about 450 ng / mL, about 460 ng / mL, about 470 ng / mL, about 480 ng / mL, about 490 ng / mL, about 500 ng / mL, about 5 The OKT3 antibody may be present at a concentration of 000ng / mL, about 200ng / mL, about 500ng / mL, about 1µg / mL, or between 0.1ng / mL and 1ng / mL, between 1ng / mL and 5ng / mL, between 5ng / mL and 10ng / mL, between 10ng / mL and 20ng / mL, between 20ng / mL and 30ng / mL, between 30ng / mL and 40ng / mL, 40ng / mL and 50ng / mL, or 50ng / mL and 100ng / mL.

[0165] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is utilized as a combination during expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during expansion. In some embodiments, a combination of IL-2, IL-15, and IL-21 is utilized as a combination during expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof may be included. In some embodiments, expansion may be performed in supplemented cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells.

[0166] In some embodiments, the expansion culture medium contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 1 00 IU / mL IL-15, or about 500 IU / mL to about 100 IU / mL IL-15, or about 400 IU / mL to about 100 IU / mL IL-15, or about 300 IU / mL to about 100 IU / mL IL-15, or about 200 IU / mL IL-15, or about 180 IU / mL IL-15.

[0167] In some embodiments, the expansion culture medium contains about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21, or about 2 IU / mL of IL-21. The cell culture medium contains 0 IU / mL to about 0.5 IU / mL of IL-21, or about 15 IU / mL to about 0.5 IU / mL of IL-21, or about 12 IU / mL to about 0.5 IU / mL of IL-21, or about 10 IU / mL to about 0.5 IU / mL of IL-21, or about 5 IU / mL to about 1 IU / mL of IL-21, or about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21 or about 0.5 IU / mL of IL-21. In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of CoStAR cells to PBMCs and / or antigen-presenting cells during expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500, or between 1:50 and 1:300, or between 1:100 and 1:200.

[0168] In certain embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or can be linked to a surface. If surface-linked, the agents can be linked to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be linked to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or linked to a surface. In certain embodiments, both agents can be in solution. In one embodiment, the agents can be in soluble form and then cross-linked to a surface, a cell expressing an Fc receptor, or an antibody or other binding agent that binds to the agent, etc. In this regard, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) contemplated for use in the present invention in activating and expanding T cells.

[0169] In one embodiment, the two types of agents are immobilized on beads, either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, both of which are co-immobilized on the same bead with comparable molecular weights. In one embodiment, the respective antibodies bound to the beads for CD4+ T cell expansion and T cell proliferation are used in a 1:1 ratio. In certain embodiments of the invention, a ratio of anti-CD3 antibody:anti-CD28 antibody bound to the beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one particular embodiment, an increase of about 1-fold to about 3-fold is observed compared to the expansion observed using a 1:1 ratio. In one embodiment, the ratio of CD3 antibody:CD28 antibody bound to the beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one embodiment of the invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2:1. In one particular embodiment, antibodies bound to beads at a CD3:CD28 ratio of 1:100 are used. In one embodiment, antibodies bound to beads at a CD3:CD28 ratio of 1:75 are used. In a further embodiment, antibodies bound to beads at a CD3:CD28 ratio of 1:50 are used. In one embodiment, antibodies bound to beads at a CD3:CD28 ratio of 1:30 are used. In a preferred embodiment, antibodies bound to beads at a CD3:CD28 ratio of 1:10 are used. In one embodiment, antibodies bound to beads at a CD3:CD28 ratio of 1:3 are used. In a further embodiment, antibodies bound to beads at a CD3:CD28 ratio of 3:1 are used.

[0170] To stimulate T cells and other target cells, particle-to-cell ratios ranging from 1:500 to 500:1, and any integer value therebetween, can be used. As one skilled in the art can readily appreciate, the particle-to-cell ratio can depend on the size of the particles relative to the target cells. For example, small beads can only bind a few cells, while larger beads can bind many. In certain embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1, and any integer value therebetween, and in further embodiments, the ratio includes 1:9 to 9:1, and any integer value therebetween, and can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells resulting in T cell stimulation can vary as described above; however, certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 particles per T cell. In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In one particular embodiment, a preferred particle:cell ratio is 1:5. In further embodiments, the particle-to-cell ratio can vary depending on the day of stimulation. For example, in one embodiment, the particle to cell ratio is 1:1 to 10:1 on the first day, and then additional particles are added to the cells every day or every other day for up to 10 days, with a final ratio of 1:1 to 1:10 (based on cell count on the day of addition). In one particular embodiment, the particle to cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In one embodiment, particles are added every day or every other day, with a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In one embodiment, the particle to cell ratio is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one embodiment, particles are added every day or every other day, with a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation.Those skilled in the art will understand that various other ratios may be suitable for use in the present invention. In particular, the ratio will vary depending on the size of the particles and the size and type of cells. In one embodiment, the most typical ratios to use are around 1:1, 2:1, and 3:1 on the first day.

[0171] In a further embodiment of the invention, cells, e.g., T cells, are combined with agent-coated beads, and then the beads and cells are separated before culturing the cells. In an alternative embodiment, the agent-coated beads and cells are not separated but are cultured together prior to culturing. In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation. Preparation of CoStAR cells Viral and non-viral genetic engineering tools can be used to generate CoStAR cells, including but not limited to T cells and NK cells, that allow for permanent or transient expression of therapeutic genes. Retroviral gene delivery is a mature and well-characterized technology that has been used to permanently integrate CARs into the host cell genome (Scholler J., e.g., Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med. 2012;4:132ra53; Rosenberg SA et al., Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 1990;323:570-578).

[0172] Non-viral DNA transfection methods can also be used.For example, Singh et al. have described the use of the Sleeping Beauty (SB) transposon system developed to engineer CAR T cells (Singh H., et al., Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res. 2008;68:2961-2971), and has been used in clinical trials (see, for example, ClinicalTrials.gov:NCT00968760 and NCT01653717).The same technology can also be applied to engineer CoStAR cells.

[0173] Several SB enzymes have been used to deliver transgenes. Mates described a hyperactive transposase (SB100X) that exhibits approximately 100-fold enhanced efficiency compared to first-generation transposases. SB100X supports stable gene transfer in 35-50% of human CD34(+) cells enriched for hematopoietic stem or progenitor cells (Mates L. et al., Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 2009;41:753-761), and multiple transgenes can be delivered in a multicistronic single plasmid (e.g., Thokala R. et al., Redirecting specificity of T cells using the Sleeping Beauty system to express chimeric antigen receptors by mix-and-matching of VL and VH domains targeting CD123+ tumors. PLoS ONE. 2016;11:e0159477) or multiple plasmids (e.g., Hurton LV et al., Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc. Natl. Acad. Sci. USA. 2016;113:E7788-E7797). Such systems can be used with CoStAR of the present invention.

[0174] Morita et al. described the piggyBac transposon system for integrating larger transgenes (Morita D. et al., Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol. Ther. Methods Clin. Dev. 2017;8:131-140), and Nakazawa et al. described the use of this system to generate EBV-specific cytotoxic T cells expressing a HER2-specific chimeric antigen receptor (Nakazawa Y et al., PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther. 2011;19:2133-2143). Manuri et al. used this system to generate CD19-specific T cells (Manuri PVR et al., piggyBac transposon / transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum. Gene Ther. 2010;21:427-437).

[0175] Transposon technology is easy and economical. One potential drawback is that the currently used longer expansion protocols can lead to T cell differentiation, loss of activity, and poor persistence of injected cells. Monjezi et al. have described the development of minicircle vectors that minimize these problems through more efficient integration (Monjezi R. et al., Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia. 2017;31:186-194). These transposon technologies can be used for CoStAR of the present invention. Pharmaceutical Composition The present invention also relates to pharmaceutical compositions comprising a vector or a CoStAR-expressing cell of the present invention together with a pharmaceutically acceptable carrier, diluent, or excipient, and optionally one or more additional pharmaceutically active polypeptides and / or compounds.

[0176] In some embodiments, a pharmaceutical composition is provided, comprising the above-mentioned CoStAR and a pharmaceutically acceptable carrier.In some embodiments, a pharmaceutical composition is provided, comprising the nucleic acid encoding CoStAR according to any of the above-mentioned embodiments and a pharmaceutically acceptable carrier.In some embodiments, a pharmaceutical composition is provided, comprising the above-mentioned effector cell expressing CoStAR and a pharmaceutically acceptable carrier.Such a formulation can be, for example, in a form suitable for intravenous infusion. As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components in the composition in which it is contained. A pharmaceutically acceptable carrier or excipient preferably meets the required standards of toxicology and manufacturing testing and / or is included in the Inactive Ingredient Guide prepared by the US Food and Drug Administration.

[0177] An embodiment of the present invention provides a modified T cell population that expresses recombinant CoStAR. Suitable populations can be produced by the methods described above. The modified T cell population can be used as a medicine. For example, the modified T cell population described herein can be used in cancer immunotherapy, such as adoptive T cell therapy. Other aspects of the invention provide uses of the modified T cell populations described herein for the manufacture of a medicament for the treatment of cancer, the modified T cell populations described herein for the treatment of cancer, and methods of treating cancer that may include administering the modified T cell populations described herein to an individual in need thereof. The engineered T cell population may be autologous, i.e., the engineered T cells were originally obtained from the same individual to which they are later administered (i.e., the donor and recipient individuals are the same). An engineered T cell population suitable for administration to an individual may be produced by a method comprising providing an initial T cell population obtained from the individual, engineering the T cells to express a cAMP PDE or a fragment thereof and an antigen receptor that specifically binds to cancer cells in the individual, and culturing the engineered T cells.

[0178] The engineered T cell population may be allogeneic, i.e., the engineered T cells were originally obtained from an individual different from the individual to whom they will subsequently be administered (i.e., the donor and recipient individuals are different). The donor and recipient individuals may be HLA-matched to avoid GVHD and other undesirable immune effects. An engineered T cell population suitable for administration to a recipient individual may be produced by a method comprising providing an initial T cell population obtained from a donor individual, engineering the T cells to express CoStAR that specifically binds to cancer cells in the recipient individual, and culturing the engineered T cells. Following administration of the modified T cells, the recipient individual may mount a T cell-mediated immune response against cancer cells in the recipient individual, which may have a beneficial effect on the cancerous condition in the individual.

[0179] Cancerous conditions may be characterized by the abnormal proliferation of malignant cancer cells, and may include leukemias such as AML, CML, ALL, and CLL, lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, and solid cancers such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gallbladder and bile duct, thyroid cancer, thymus cancer, bone cancer, and cerebral cancer, and cancer of unknown primary site (CUP). Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual (i.e., cancerous tumors may be immunogenic). For example, cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells. The tumor antigens that elicit the immune response may be specific to the cancer cells or may be shared with one or more normal cells in the individual.

[0180] An individual suitable for the above-described treatment may be a mammal, such as a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, primate (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human. In preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals may be utilized, particularly mammals traditionally used as models for therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or lagomorph animals).

[0181] Treatment method The term "therapeutically effective amount" refers to an amount of a CoStAR or a composition comprising a CoStAR disclosed herein effective to "treat" a disease or disorder in an individual. In the case of cancer, a therapeutically effective amount of a CoStAR or a composition comprising a CoStAR disclosed herein can reduce the number of cancer cells, reduce the size or weight of a tumor, inhibit (i.e., slow to some extent, and preferably stop) the infiltration of cancer cells into surrounding organs, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent a CoStAR or a composition comprising a CoStAR disclosed herein can prevent the growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. In some embodiments, a therapeutically effective amount is a growth-inhibitory amount. In some embodiments, a therapeutically effective amount is an amount that improves a patient's progression-free survival. In the case of an infectious disease, e.g., a viral infection, a therapeutically effective amount of CoStAR or a composition comprising CoStAR disclosed herein can reduce the number of cells infected with a pathogen, reduce the production or release of antigens derived from the pathogen, inhibit (i.e., slow to some extent, and preferably stop) the spread of the pathogen to uninfected cells, and / or alleviate to some extent one or more symptoms associated with the infection. In some embodiments, a therapeutically effective amount is an amount that extends the survival of a patient.

[0182] Cells expressing CoStAR for use in the methods of the present invention, including T cells and NK cells, can be generated ex vivo either from the patient's own peripheral blood (autologous), from donor peripheral blood in the context of hematopoietic stem cell transplantation (allogeneic), or from peripheral blood derived from an unrelated donor (allogeneic). Alternatively, T cells or NK cells can be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into T cells or NK cells. In these examples, T cells expressing CoStAR and, optionally, CAR and / or TCR are generated by introducing DNA or RNA encoding CoStAR, and optionally, CAR and / or TCR, by one of a number of means, including viral vector transduction, DNA or RNA transfection. T cells or NK cells expressing the CoStAR of the present invention and optionally expressing a TCR and / or a CAR can be used to treat hematopoietic cancers or solid tumors.

[0183] Methods for treating diseases involve the therapeutic use of vectors or cells, including T cells or NK cells, of the present invention. In this regard, the vectors or T cells or NK cells can be administered to a subject with an existing disease or condition to alleviate, reduce, or ameliorate at least one symptom associated with the disease and / or to delay, reduce, or block the progression of the disease. The methods of the present invention can cause or promote T cell-mediated killing of cancer cells. The vectors, T cells, or NK cells of the present invention can be administered to a patient together with one or more additional therapeutic agents. The one or more additional therapeutic agents can be co-administered to a patient. "Co-administering" means administering one or more additional therapeutic agents and the vectors or T cells or NK cells of the present invention sufficiently closely in time that the vectors or T cells or NK cells can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the vectors or cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the vector or cells and one or more additional therapeutic agents may be administered simultaneously. One co-administered therapeutic agent that may be useful is IL-2, as it is currently used in existing cell therapies to boost the activity of the administered cells. However, IL-2 treatment is associated with toxicity and tolerance issues.

[0184] As described, for administration to a patient, the CoStAR effector cells may be allogeneic or autologous to the patient. In some embodiments, the allogeneic cells are further genetically modified, e.g., by gene editing, to minimize or prevent GVHD and / or immune responses in the patient against the CoStAR cells. CoStAR effector cells are used to treat cancers and neoplastic diseases associated with target antigens.Cancers and neoplastic diseases that can be treated using any of the methods described herein include non-vascularized tumors or tumors that have not yet been substantially vascularized, and vascularized tumors.Cancers may include non-solid tumors (e.g., hematological tumors, such as leukemia and lymphoma) or solid tumors.The types of cancers that can be treated with the CoStAR effector cells of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphatic malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0185] Hematological cancer is cancer of blood or bone marrow.Examples of hematological (or hematopoietic) cancer include leukemia, for example, acute leukemia (for example, acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (for example, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (late-onset and aggressive forms), multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0186] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, e.g., sarcomas and carcinomas, include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, gastric cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullary thyroid carcinoma and papillary thyroid carcinoma), pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma carcinoma), choriomas, Wilms' tumor, cervical cancer (e.g., cervical carcinoma and preinvasive cervical dysplasia), colorectal cancer, cancer of the anus, cancer of the anal canal or anorectal cancer, vaginal cancer, cancer of the vulva (e.g., squamous cell carcinoma, carcinoma in situ, adenocarcinoma, and fibrosarcoma), penile cancer, oropharyngeal cancer, esophageal cancer, head cancer (e.g., squamous cell carcinoma), cervical cancer (e.g., squamous cell carcinoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriomas, sarcomas, Leydig cell tumors, fibromas, fibroadenomas, adenocarcinomas, and sebaceous tumors), lipoma), bladder cancer, kidney cancer, melanoma, uterine cancer (e.g., endometrial cancer), urothelial cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral cancer, and bladder cancer), and CNS tumors (e.g., glioma (e.g., brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0187] When an "immunologically effective amount," "antitumor effective amount," "tumor-inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). The pharmaceutical compositions comprising the T cells described herein can be administered in an amount of 10 4 ~10 9 The cells may be administered at a dosage of 10 cells / kg of body weight, and in some instances, 10 5 ~10 6 The T cell compositions may be administered in doses of up to 1000 cells / kg of body weight, and may generally be stated to include all integers within these ranges. The T cell compositions may also be administered multiple times at these dosages. The cells may be administered using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0188] Combination therapy The CoStAR-expressing cells described herein can be used in combination with other known drugs and therapeutic agents. As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject during the course of a disease associated with the subject's disorder, for example, two or more treatments are delivered after the subject is diagnosed with the disorder and before the disorder is cured or eliminated, or before the treatment is terminated for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, so there is overlap in terms of administration. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In either case, in some embodiments, the treatments are more effective due to the combined administration. For example, the second treatment may be more effective, e.g., a comparable effect may be observed with less of the second treatment, or the second treatment may reduce symptoms to a greater extent than would be observed if the second treatment were administered without the first treatment, or if a similar situation were observed with the first treatment. In some embodiments, delivery is such that the reduction in symptoms, or other parameters associated with the disorder, is greater than would be observed if one treatment were delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may also be such that the effect of the first treatment being delivered is still detectable when the second is delivered.

[0189] The CoStAR-expressing cells described herein and at least one additional therapeutic agent may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the CAR-expressing cells described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed. CoStAR therapy and / or other therapeutic agents, procedures, or modalities can be administered during periods when the disorder is active or during periods of remission or low disease activity. CoStAR therapy can be administered prior to other treatments, concurrently with treatment for the disorder, after treatment, or during remission.

[0190] When administered in combination, the therapeutic agent and / or additional agent (e.g., a second or third agent) can be administered in amounts or dosages that are higher, lower, or the same as the amount or dosage of each agent used individually, e.g., as a monotherapy. In some embodiments, the administered amount or dosage of the CoStAR therapy, additional agent (e.g., a second or third agent), or all of these is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the CoStAR therapy, additional agent (e.g., a second or third agent), or all of these that produces a desired effect (e.g., cancer treatment) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent required individually, e.g., as a monotherapy, to achieve the same therapeutic effect.

[0191] In further embodiments, the CoStAR-expressing cells described herein can be used in treatment regimens in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapeutics, cytoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation, peptide vaccines such as those described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0192] In certain instances, the compounds of the present invention are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergy agents, anti-emetic (or anti-nausea) agents, pain relieving agents, cytoprotective agents, and combinations thereof. In one embodiment, the CoStAR-expressing cells described herein can be used in combination with chemotherapeutic agents.Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab). , antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0193] Common chemotherapy agents that may be considered for use in combination therapy include busulfan (Myleran®), busulfan injection (Busulfex®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®), daunorubicin hydrochloride (Cerubidine®), and daunorubicin hydrochloride (Cerubidine®). These include bicin citrate liposomal injection (DaunoXome®), dexamethasone, doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), idarubicin (Idamycin®), mitoxantrone (Novantrone®), and gemtuzumab ozogamicin (mylotarg®).

[0194] In embodiments, common chemotherapeutic agents contemplated for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentaoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine ( BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitidine, gemcitabine ( difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg,These include paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0195] Treatment can be evaluated, for example, by tumor regression, reduction in tumor mass or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Approaches to determining the efficacy of therapy can be utilized, including, for example, measuring response through radiological imaging.

[0196] array The following sequences include, but are not limited to, the complete CoStAR and CoStAR components. The components include the signal peptide (SP), binding domain (BD), linker, spacer, and transmembrane domain (STM), the CD28 transmembrane fragment without extracellular or intracellular sequences (STM-CD28TM), the intracellular signal domain (SD), and the CD40 domain and motif. While SEQ ID NOs: 42-247 include CoStAR with an N-terminal signal peptide, it will be understood that the N-terminal signal peptide is removed from mature CoStAR. Furthermore, it will be understood that mature CoStAR may lack one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids at the N-terminus (i.e., counting from the C-terminus of the signal peptide). The location of components within the entire protein can be confirmed from GenBank and other sources. The constructs and components are exemplary with respect to exact size, and the range and components may be derived from more than one source. When more than one intracellular signaling domain or signaling fragment is present, the domains can be in any order. It will be understood that while certain proteins may contain N-terminal signal peptides when expressed, these signal peptides can be cleaved or cleaved incorrectly when the protein is expressed, and that removal of the signal peptide results in a protein containing a binding domain with a variation of up to about five amino acids at the N-terminal amino acid position.

[0197] [Table 7] TIFF2025508319000009.tif224169 TIFF2025508319000010.tif195169 TIFF2025508319000011.tif227169 TIFF2025508319000012.tif233169 TIFF2025508319000013.tif212169 TIFF2025508319000014.tif201169 TIFF2025508319000015.tif191169 TIFF2025508319000016.tif191169 TIFF2025508319000017.tif238169 TIFF2025508319000018.tif238169 TIFF2025508319000019.tif217169 TIFF2025508319000020.tif201169 TIFF2025508319000021.tif185169 TIFF2025508319000022.tif191169 TIFF2025508319000023.tif238169 TIFF2025508319000024.tif169169 TIFF2025508319000025.tif227169 TIFF2025508319000026.tif212169 TIFF2025508319000027.tif206169 TIFF2025508319000028.tif196169 TIFF2025508319000029.tif227169 TIFF2025508319000030.tif233169 TIFF2025508319000031.tif217169 TIFF2025508319000032.tif206169 TIFF2025508319000033.tif206169 TIFF2025508319000034.tif196169 TIFF2025508319000035.tif185169 TIFF2025508319000036.tif185169 TIFF2025508319000037.tif206169 TIFF2025508319000038.tif227169 TIFF2025508319000039.tif227169 TIFF2025508319000040.tif217169 TIFF2025508319000041.tif206169 TIFF2025508319000042.tif206169 TIFF2025508319000043.tif196169 TIFF2025508319000044.tif185169 TIFF2025508319000045.tif180169 TIFF2025508319000046.tif211169 TIFF2025508319000047.tif227169 TIFF2025508319000048.tif227169 TIFF2025508319000049.tif217169 TIFF2025508319000050.tif206169 TIFF2025508319000051.tif206169 TIFF2025508319000052.tif196169 TIFF2025508319000053.tif185169 TIFF2025508319000054.tif185169 TIFF2025508319000055.tif185169 TIFF2025508319000056.tif185169 TIFF2025508319000057.tif185169 TIFF2025508319000058.tif185169 TIFF2025508319000059.tif185169 TIFF2025508319000060.tif185169 TIFF2025508319000061.tif185169 TIFF2025508319000062.tif185169 TIFF2025508319000063.tif185169 TIFF2025508319000064.tif185169 TIFF2025508319000065.tif185169 TIFF2025508319000066.tif185169 TIFF2025508319000067.tif185169 TIFF2025508319000068.tif185169 TIFF2025508319000069.tif185169 TIFF2025508319000070.tif185169 TIFF2025508319000071.tif185169 TIFF2025508319000072.tif185169 TIFF2025508319000073.tif233169 TIFF2025508319000074.tif164169 TIFF2025508319000075.tif227169 TIFF2025508319000076.tif227169 TIFF2025508319000077.tif227169 TIFF2025508319000078.tif206169 TIFF2025508319000079.tif206169 TIFF2025508319000080.tif206169 TIFF2025508319000081.tif180169 TIFF2025508319000082.tif180169 TIFF2025508319000083.tif175169 TIFF2025508319000084.tif227169 TIFF2025508319000085.tif227169 TIFF2025508319000086.tif227169 TIFF2025508319000087.tif206169 TIFF2025508319000088.tif206169 TIFF2025508319000089.tif201169 TIFF2025508319000090.tif175169 TIFF2025508319000091.tif175169 TIFF2025508319000092.tif169169 TIFF2025508319000093.tif227169 TIFF2025508319000094.tif227169 TIFF2025508319000095.tif227169 TIFF2025508319000096.tif206169 TIFF2025508319000097.tif206169 TIFF2025508319000098.tif206169 TIFF2025508319000099.tif206169 TIFF2025508319000100.tif180169 TIFF2025508319000101.tif175169 TIFF2025508319000102.tif227169 TIFF2025508319000103.tif227169 TIFF2025508319000104.tif227169 TIFF2025508319000105.tif206169 TIFF2025508319000106.tif206169 TIFF2025508319000107.tif206169 TIFF2025508319000108.tif185169 TIFF2025508319000109.tif180169 TIFF2025508319000110.tif175169 TIFF2025508319000111.tif227169 TIFF2025508319000112.tif227169 TIFF2025508319000113.tif227169 TIFF2025508319000114.tif206169 TIFF2025508319000115.tif206169 TIFF2025508319000116.tif206169 TIFF2025508319000117.tif185169 TIFF2025508319000118.tif185169 TIFF2025508319000119.tif185169 TIFF2025508319000120.tif232169 TIFF2025508319000121.tif232169 TIFF2025508319000122.tif227169 TIFF2025508319000123.tif206169 TIFF2025508319000124.tif206169 TIFF2025508319000125.tif206169 TIFF2025508319000126.tif185169 TIFF2025508319000127.tif185169 TIFF2025508319000128.tif185169 TIFF2025508319000129.tif185169 TIFF2025508319000130.tif185169 TIFF2025508319000131.tif185169 TIFF2025508319000132.tif185169 TIFF2025508319000133.tif185169 TIFF2025508319000134.tif185169 TIFF2025508319000135.tif185169 TIFF2025508319000136.tif185169 TIFF2025508319000137.tif185169 TIFF2025508319000138.tif185169 TIFF2025508319000139.tif185169 TIFF2025508319000140.tif185169 TIFF2025508319000141.tif185169 TIFF2025508319000142.tif185169 TIFF2025508319000143.tif185169 TIFF2025508319000144.tif185169 TIFF2025508319000145.tif185169 TIFF2025508319000146.tif185169 TIFF2025508319000147.tif228169 TIFF2025508319000148.tif186169 TIFF2025508319000149.tif190169 TIFF2025508319000150.tif180169 TIFF2025508319000151.tif195169 TIFF2025508319000152.tif227169 TIFF2025508319000153.tif127169 TIFF2025508319000154.tif148169 TIFF2025508319000155.tif127169 TIFF2025508319000156.tif148169 TIFF2025508319000157.tif232169 TIFF2025508319000158.tif227169 TIFF2025508319000159.tif232169 TIFF2025508319000160.tif180169 TIFF2025508319000161.tif227169 TIFF2025508319000162.tif227169 TIFF2025508319000163.tif238169 TIFF2025508319000164.tif222169 TIFF2025508319000165.tif227169 TIFF2025508319000166.tif138169 TIFF2025508319000167.tif238169 TIFF2025508319000168.tif217169 TIFF2025508319000169.tif206169 TIFF2025508319000170.tif217169 TIFF2025508319000171.tif211169 TIFF2025508319000172.tif138169 TIFF2025508319000173.tif132169 TIFF2025508319000174.tif132169 TIFF2025508319000175.tif138169 TIFF2025508319000176.tif159169 TIFF2025508319000177.tif148169 TIFF2025508319000178.tif138169 TIFF2025508319000179.tif148169 TIFF2025508319000180.tif148169 TIFF2025508319000181.tif132169 TIFF2025508319000182.tif127169 TIFF2025508319000183.tif227169 TIFF2025508319000184.tif217169 TIFF2025508319000185.tif227169 TIFF2025508319000186.tif117169 TIFF2025508319000187.tif138169 TIFF2025508319000188.tif238169 TIFF2025508319000189.tif227169 TIFF2025508319000190.tif196169 TIFF2025508319000191.tif217169 ...

Claims

1. Consensus A sequence or X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 A protein comprising an amino acid sequence comprising: a.X 1 is any amino acid, b. X 2 is any amino acid, C. X 3 is P and X 4 is any amino acid, d. X 5 is Q, e. X 6 is C or A, f.X 7 is any amino acid, g. X 8 is any amino acid, h.X 9 is any amino acid, i.X 10 is any amino acid, j.X 11 is any amino acid, The protein.

2. The protein of claim 1 which is part of a TRAF domain.

3. 3. A protein according to claim 1 or 2, which is part of the TRAF2 domain.

4. a. The sequence is i.X 1 is S or P, ii. X 2 is V, H, or Y; iii. X 4 are I, Q, and V, iv. X 7 is T or S, v.X 8 is D, vi. X 9 is K, D, or G; vii. X 10 is T, S, G, or A; viii. X 11 is D, S, N, or D Contains a sequence, or b. the sequence is at least 60% identical to a); The protein according to any one of claims 1 to 3.

5. Consensus B sequence or X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 A protein comprising an amino acid sequence comprising: a.X 1 is any amino acid, b. X 2 is any amino acid, C. X 3 is P, S, A, or T; d. X 4 is any amino acid, e. X 5 is Q or E, f.X 6 is E, g. X 7 is any amino acid, h.X 8 is any amino acid, i.X 9 is any amino acid, j.X 10 is any amino acid, K.X 11 is any amino acid, The protein.

6. The protein of claim 5 which is part of a TRAF domain.

7. 7. A protein according to claim 5 or 6, which is part of the TRAF2 domain.

8. a. The sequence is i.X 1 are P, A, M, T, S, R, V, and H; ii. X 2 are F, A, L, I, T, V, G, C, A, F, and P; iii. X 4 are K, V, I, H, A, Q, T, E, S, iv. X 7 are C, T, E, D, S, and A; v.X 8 are A, L, G, Y, I, Q, D, and E, vi. X 9 are F, H, K, R, P, A, G, Y, E, and N; vii. X 10 are R, G, E, K, A, S, D, C, C, P, V, viii. X 11 are S, C, D, P, W, T, A, G, and E. Contains a sequence, or b. the sequence is at least 60% identical to a); The protein according to any one of claims 5 to 7.

9. The protein according to any one of claims 1 to 8, wherein the sequence is a part of the Traf2 / 3 sequence.

10. Consensus C sequence or X 1 X 2 X 3 X 4 X 5 X 6 A protein comprising an amino acid sequence comprising: a.X 1 is P, b. X 2 is any amino acid, C. X 3 is E, d. X 4 is any amino acid, e. X 5 is any amino acid, f.X 6 is Ac / Ar; The protein.

11. The protein of claim 5 which is part of a TRAF domain.

12. The protein according to any one of claims 1 to 11, which is a part of the TRAF6 domain.

13. a. The sequence is i.X 2 is Q, P, E, V, T, or S; ii. X 4 are I, L, M, N, S, T, N, D, iii. X 5 is N, D, R, S, G, or Y Contains a sequence, or b. the sequence is at least 60% identical to a); The protein according to any one of claims 10 to 12.

14. d) one or more of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; e) a sequence that is at least 70% identical to the sequence in a); or f) a sequence that is at least 80% similar to a). An amino acid sequence comprising:

15. A TRAF domain in a CD40 protein, said TRAF domain comprising: d) SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; e) a sequence that is at least 70% identical to the sequence in a); or f) a sequence that is at least 80% similar to a). The TRAF domain comprising one or more of:

16. TRAF6 domain sequences including PQEINF, PEEMSW, PPENYE, or PQENSY.

17. A TRAF2 / 3 domain sequence comprising PVQET, TQEET, SKEET, AVEET, or PVQET.

18. A TRAF2 domain sequence including SVQE, AVEE, or PEEE.

19. d) the amino acid sequence of one of SEQ ID NOs: 475, 476, 477, 478, 479, 494, 495, 496, 498, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, or 519; e) an amino acid sequence that is at least 70% identical to a); or f) an amino acid sequence that is at least 80% similar to a); A TRAF6 or TRAF2 / 3 domain comprising:

20. 20. The protein, amino acid sequence or TRAF domain of any one of claims 1 to 19, wherein the sequence is within the CD40 protein sequence.

21. 21. The protein, amino acid sequence or TRAF domain of any one of claims 1 to 20, wherein the sequence is within the CD40 protein and maps to the corresponding TRAF2, TRAF2 / 3 and / or TRAF6 domain within said CD40 protein.

22. Consensus A or X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 A TRAF2 / TRAF3 domain comprising the amino acid sequence of a.X 1 is any amino acid, b. X 2 is any amino acid, C. X 3 is P and X 4 is any amino acid, d. X 5 is Q, e. X 6 is C or A, f.X 7 is any amino acid, g. X 8 is any amino acid, h.X 9 is any amino acid, i.X 10 is any amino acid, j.X 11 is any amino acid, The TRAF2 / TRAF3 domain.

23. Consensus B or X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 A TRAF2 / TRAF3 domain comprising the amino acid sequence of a.X 1 is any amino acid, b. X 2 is any amino acid, C. X 3 is P, S, A, or T; d. X 4 is any amino acid, e. X 5 is Q or E, f.X 6 is E, g. X 7 is any amino acid, h.X 8 is any amino acid, i.X 9 is any amino acid, j.X 10 is any amino acid, K.X 11 is any amino acid, The TRAF2 / TRAF3 domain.

24. Consensus C or X 1 X 2 X 3 X 4 X 5 X 6 A TRAF6 domain comprising the amino acid sequence a.X 1 is P, b. X 2 is any amino acid, C. X 3 is E, d. X 4 is any amino acid, e. X 5 is any amino acid, f.X 6 is Ac / Ar; The TRAF6 domain.

25. below: a. Consensus A or X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 A TRAF2 / TRAF3 domain comprising the amino acid sequence of i.X 1 is any amino acid, ii. X 2 is any amino acid, iii. X 3 is P and X 4 is any amino acid, iv. X 5 is Q, v.X 6 is C or A, vi. X 7 is any amino acid, vii. X 8 is any amino acid, viii. X 9 is any amino acid, ix. X 10 is any amino acid, x.X 11 is any amino acid, TRAF2 / TRAF3 domain, b. Consensus B or X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 A TRAF2 / TRAF3 domain comprising the amino acid sequence of i.X 1 is any amino acid, ii. X 2 is any amino acid, iii. X 3 is P, S, A, or T; iv. X 4 is any amino acid, v.X 5 is Q or E, vi. X 6 is E, vii. X 7 is any amino acid, viii. X 8 is any amino acid, ix. X 9 is any amino acid, x.X 10 is any amino acid, xi.X 11 is any amino acid, TRAF2 / TRAF3 domain, c. Consensus C or X 1 X 2 X 3 X 4 X 5 X 6 A TRAF6 domain comprising the amino acid sequence i.X 1 is P, ii. X 2 is any amino acid, iii. X 3 is E, iv. X 4 is any amino acid, v.X 5 is any amino acid, vi. X 6 is Ac / Ar; TRAF6 domain A CD40 domain comprising one or more amino acid sequences of:

26. an extracellular binding domain that binds to carcinoembryonic antigen (CEA) or an extracellular binding domain that binds to mesothelin (MSLN); a transmembrane domain linked to the extracellular binding domain; a first signaling domain; and a second signaling domain, the second signaling domain comprising a variant CD40 signaling domain or a signaling fragment thereof, the variant CD40 comprising a variant TRAF2 / TRAF3 sequence, or a variant TRAF6 sequence, or a variant TRAF2 sequence, the variant CD40 not comprising SEQ ID NO: 42; A chimeric costimulatory antigen receptor (CoStAR) comprising:

27. 27. The CoStAR of claim 26, wherein the first signaling domain comprises a signaling domain or fragment of CD28 or CD278 (ICOS).

28. 27. The CoStAR of claim 26, wherein the first signaling domain comprises a full-length costimulatory domain.

29. 27. The CoStAR of claim 26, wherein the extracellular binding domain is operably linked to the transmembrane domain by a linker and / or spacer.

30. 30. The CoStAR of claim 29, wherein the linker comprises from about 5 to about 20 amino acids.

31. 30. The CoStAR of claim 29, wherein the linker comprises from about 10 to about 250 amino acids.

32. 30. The CoStAR of claim 29, wherein the transmembrane domain comprises a transmembrane domain derived from CD28, CD8, ICOS, DAP10, or NTRK.

33. 30. The CoStAR of claim 29, wherein the transmembrane domain comprises the transmembrane domain sequence of SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

34. 30. The CoStAR of claim 29, wherein the extracellular binding domain comprises an scFv, a peptide, an antibody heavy chain variable domain, an antibody light chain variable domain, or a CEA ligand.

35. A nucleic acid encoding the CoStAR of any one of claims 1 to 34.

36. A vector comprising the nucleic acid of claim 35.

37. A cell expressing CoStAR according to any one of claims 1 to 34.

38. 38. The cell of claim 37, comprising an alpha-beta T cell, a gamma-delta T cell, a regulatory T cell, a TIL, a NKT cell, or a NK cell.

39. The cell of claim 37, which co-expresses a CAR or a TCR.

40. A method of producing a cell according to claims 37 to 39, comprising the step of transducing or transfecting a cell with a vector according to claim 36.

41. A method for preparing a cell population expressing CoStAR according to any one of claims 26 to 34, comprising: a. detecting expression of CoStAR on the surface of a cell transfected or transduced with the vector of claim 36; b. Selecting cells identified as expressing CoStAR; Including, The method.

42. A cell population enriched for cellular expression of CoStAR according to any one of claims 26 to 34.

43. 43. A method for treating a disease in a subject, comprising administering to the subject a cell according to any one of claims 37 to 39 or a cell population according to claim 42.

44. A protein comprising one of the amino acid sequences of SEQ ID NOs:510-519.

45. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

510.

46. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

511.

47. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

512.

48. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

513.

49. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

514.

50. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

515.

51. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

516.

52. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

517.

53. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:

518.

54. 45. The protein of claim 44, comprising the sequence of SEQ ID NO:519.

Images