Receptors providing targeted co-stimulation for adoptive cell therapy

Abstract

The present invention relates to chimeric co-stimulatory antigen receptors (CoStAR) useful in adoptive cell therapy (ACT) and cells comprising said CoStAR. CoStAR can be used as modulators that enhance the activity of cells that respond to determined antigens. The invention also provides CoStAR proteins, nucleic acids encoding said CoStAR, and therapeutic uses thereof.

Description

Related Applications and Incorporation by Reference

[0001] This application is a continuation in part of U.S. application No. 17 / 807109, filed on June 15, 2022, entitled “RECEPTORS PROVIDING TARGETED COSTIMULATION FOR ADOPTIVE CELL THERAPY,” and both this application and 17 / 807109 claim priority to U.S. provisional application No. 63 / 301340, filed on January 20, 2022, entitled “RECEPTORS PROVIDING TARGETED COSTIMULATION FOR ADOPTIVE CELL THERAPY,” each of which is incorporated by reference in its entirety.

[0002] Reference is made to U.S. provisional patent applications 63 / 211,042 and 63 / 211,046 filed on June 16, 2021.

[0003] Reference is made to UK patent application serial number 1900858.0 filed on January 22, 2019, U.S. patent application serial number 62 / 951,770 filed on December 20, 2019, international application PCT / GB2020 / 050120 filed on January 20, 2020, and U.S. provisional patent applications 63 / 053,494 and 63 / 053,498 filed on July 17, 2020.

[0004] All documents cited in the aforementioned applications and those applications or during their prosecution ("application-cited documents") and all documents cited or referenced in the application-cited documents and all documents cited or referenced herein ("herein-cited documents") and all documents cited or referenced in the herein-cited documents, as well as any manufacturer's instructions, descriptions, product data sheets, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the present 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. Reference Sequence Listing

[0005] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file titled 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. Field of the Invention

[0006] The present invention relates to variants of the TRAF domain. Background of the Invention

[0007] Adoptive cell therapy (ACT) using autologous T cells to mediate cancer regression has shown great promise in early clinical trials. Several general methods have been used, such as using naturally occurring tumor reactive or tumor infiltrating lymphocytes (TILs) amplified in vitro. In addition, T- cells can be genetically modified to retarget them to determined tumor antigens. This can be accomplished via gene transfer of peptide (p)-major histocompatibility complex (MHC)-specific T cell receptors (TCRs) or synthetic fusions between tumor-specific single-chain antibody fragments (scFv) and T cell signaling domains (such as CD3ζ), the latter being referred to as chimeric antigen receptors (CARs).

[0008] TIL and TCR transfer have been shown to be particularly effective when targeting melanoma (Rosenberg et al., 2011; Morgan 2006), while CAR therapy has shown great promise in the treatment of certain B-cell malignancies (Grupp et al., 2013).

[0009] Costimulatory signals can be used to achieve robust CAR T cell expansion, function, persistence and anti-tumor activity. The success of CAR therapy in leukemia has been partially attributed to the incorporation of costimulatory domains (such as CD28 or CD137) into CAR constructs, and the signals from these costimulatory domains synergize with the signals provided by CD3ζ to enhance anti-tumor activity. The basis of this observation is related to the classical signal 1 / signal 2 pattern of T cell activation. Here, the signal 1 provided by the TCR complex synergizes with the signal 2 provided by costimulatory receptors such as CD28, CD137 or CD134, allowing cells to undergo clonal expansion, IL2 production and long-term survival without activation-induced cell death (AICD) associated with a single signal 1. In addition, the participation of signal 2 enhances the signal generated by signal 1, allowing cells to better respond to low-affinity interactions, such as those encountered during anti-tumor responses.

[0010] Targeting co-stimulation will have beneficial effects on non-CAR-based T cell therapies. For example, incorporating co-stimulatory domains into chimeric TCRs has been shown to enhance T cell responses to pMHC (Govers 2014). In addition, there has been a trend towards generating chimeric co-stimulatory receptors that separate signal 2, which produces the co-stimulatory domain, from signal 1, which produces the CD3ζ (or other) portion, onto a separate receptor, which can also reduce the opportunity for tonic signaling (Fisher et al., 2019).

[0011] Although tumor-infiltrating lymphocytes (TILs) utilize their endogenous TCRs to mediate tumor recognition, it is not yet possible to engineer endogenous TCRs. Consequently, TILs are considerably limited because tumor cells express very few costimulatory ligands. The ability to induce targeted costimulation in TILs, or indeed any other adoptive T cell therapy products, would be beneficial.

[0012] 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

[0013] The present invention provides a new chimeric costimulatory antigen receptor (CoStAR) combined with carcinoembryonic antigen (CEA) and a cell comprising or expressing CoStAR, which is beneficial to both CAR and non-CAR based T cell therapies. The present invention uses cells expressing new chimeric costimulatory receptors to provide costimulatory signals to T- cells when combined with a determined disease-related (e.g., tumor-related) antigen.

[0014] There have been several reports in which separating signal 1 and signal 2 has been used to drive antigen-specific responses in engineered T cells (Alvarez-Vallina & Hawkins 1996). However, none of these utilized the full-length CD28 molecule. Using a full-length receptor, such as CD28, as opposed to a truncated form, has certain advantages. While the full-length receptor may be able to dimerize, allowing the receptor to function in its native form, chimeric antigen receptors may not function optimally when expressed as monomers (Bridgeman et al., 2010).

[0015] In some embodiments, the CoStAR of the present invention induces signal 2 when bound to a determined antigen (such as a disease-associated or tumor-associated antigen). The full-length CD28 molecule contains a motif that is critical to its natural function of binding to a member of the B7 receptor family; although from the perspective of a CAR carrying CD28 and CD3ζ receptors in series, connecting CAR via B7 can trigger T cell activation, which is potentially dangerous, there are beneficial properties for receptors with separate signal 2 receptors. In one aspect, the present invention provides a targeted chimeric co-stimulatory receptor (CoStAR), which comprises an extracellular binding domain operably connected to a transmembrane domain, a first signaling domain, and a CD40 signaling domain or a signaling fragment thereof. The inventors have found that co-stimulatory receptors comprising a CD40 signaling domain show new and improved activity characteristics. The inventors also found that variants of the TRAF2, TRAF2 / 3, and TRAF6 binding domains within CD40 regulate signal transduction.

[0016] 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.

[0017] In some embodiments, the first signaling domain of a CoStAR comprises a signaling domain or signaling fragment of a receptor, such as, 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, the CoStAR comprises CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD 150 (SLAM), CD270 (HVEM), CD278 (ICOS), DAP10, NTKR, CD357 (GITR), or EphB6. In embodiments, wherein the first signaling domain comprises a CD40 signaling domain, the CoStAR thus comprises elements of two CD40 signaling domains.

[0018] In some embodiments, the CoStAR comprises a second signaling domain or signaling fragment of a receptor, such as, 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 signaling fragment, the CD40 signaling domain or signaling fragment, and the second signaling domain or signaling fragment can be in any order. Exemplary embodiments include, but are not limited to, CoStARs comprising a CD28, CD137, and CD40 signaling domain, a CD28, CD134, and CD40 signaling domain, a CD28, CD2, and CD40 signaling domain, a CD28, GITR, and CD40 signaling domain, a CD28, CD29, and CD40 signaling domain, or a CD28, CD150, and CD40 signaling domain.

[0019] In some embodiments, the extracellular antigen binding domain of the CoStAR of the present invention (e.g., but not limited to, CEA binding domain, MSLN binding domain) is operably connected 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).

[0020] In some embodiments, the CoStARs of the present invention comprise a spacer region that operably connects the extracellular binding domain to the transmembrane domain and comprises about 10 to about 250 amino acids. In some embodiments, the spacer region comprises an 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 from CD8 or CD28. In some embodiments, the spacer region comprises one or more immunoglobulin domains or immunoglobulin constant regions. In some embodiments, the spacer region comprises one or more immunoglobulin domains or immunoglobulin constant regions, such as, but not limited to, SEQ ID NO: 24.

[0021] In some embodiments, the transmembrane domain of a CoStAR of the present invention comprises the transmembrane domain of a TNFRSF protein. In some embodiments, the transmembrane domain of a CoStAR of the present invention comprises the transmembrane domain of CD28 or CD8. In some embodiments, the transmembrane domain of a CoStAR of the present invention comprises the transmembrane sequence of CD28 or CD8.

[0022] The CoStARs of the present invention can be used to stimulate an immune response against a selected target expressing a tumor-associated antigen (TAA), such as, but not limited to, carcinoembryonic antigen (CEA), mesothelin (MSLN), or others. In some embodiments, the CoStAR comprises an antigen-binding fragment of a 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, such as a fragment comprising one, two, three, four, five, or all six complementarity determining regions (CDRs). In some embodiments, the CoStAR comprises a 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 CoStAR comprises an antigen-binding fragment of 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, e.g., a fragment comprising one, two, three, four, five, or all six CDRs. In some embodiments, the CoStAR comprises 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: 191.

[0023] According to the present invention, the extracellular binding domain may include, but is not limited to, a 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.

[0024] In some embodiments, a CoStAR of the invention comprises a CD3 zeta signaling domain, eg, located at the C-terminus.

[0025] In some embodiments, the CoStAR of the present invention comprises an N-terminal signal peptide. Non-limiting examples of N-terminal signal peptides are the signal peptides of oncostatin M (OSM), CD8α, CD2, interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), and human IgGκ.

[0026] In one aspect of the present invention, nucleic acids encoding CoStARs of the present invention are provided. The nucleic acids can be optimized, for example, codon optimized to express in a host cell. In a non-limiting embodiment, the nucleic acids can be codon optimized to express in human cells.

[0027] In some embodiments, vectors are provided that encode and are capable of expressing the CoStAR of any embodiment of the present disclosure.

[0028] In some embodiments, cells expressing CoStARs of any embodiment of the present disclosure are provided. In some embodiments, the cells express two or more CoStARs, for example, cells expressing CoStARs that bind to CEA or MSLN and CoStARs that bind to FolR1 or CoStARs that bind 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, cells express CoStARs that bind to CEA and CoStARs that bind to PDL1, such as, but not limited to, anti-CEA.CD28.CD40 and PD1.CD28.CD40; or cells expressing CoStARs that bind to MSLN and CoStARs that bind to PDL1, such as, but not limited to, anti-MSLN.CD28.CD40 and PD1.CD28.CD40.

[0029] In some embodiments, the cells engineered to express the CoStAR of any embodiment of the present disclosure include α-β T cells, γ-δ T cells, regulatory T cells, TIL, NKT cells or NK cells. In some embodiments, the cells engineered to express the CoStAR of any embodiment of the present disclosure co-express chimeric antigen receptor (CAR) or T cell receptor (TCR).

[0030] Some embodiments provide methods of preparing a cell expressing CoStAR comprising transducing or transfecting a cell with a vector encoding and capable of expressing the CoStAR of any embodiment of the disclosure.

[0031] Some embodiments provide methods for preparing a population of cells expressing CoStAR according to any embodiment of the present disclosure by transducing or transfecting cells, detecting expression of CoStAR, and enriching, expanding, and / or selecting cells expressing CoStAR. 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0032] Some embodiments provide methods of treating a disease in a subject by administering a cell population expressing a CoStAR according to any embodiment of the present disclosure. In some embodiments, the protein comprises a variant CD40 and / or TRAF 2, 2 / 3, 6 domain disclosed herein. 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0033] In some embodiments, the composition comprises a consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 A protein having an amino acid sequence of the sequence of , 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, X 10 is any amino acid, and X 11is 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 comprises: 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, 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 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, SEQ ID NO:42378.

[0034] In some embodiments, the composition comprises a consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 A protein having an amino acid sequence of the sequence of , 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, 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 comprises: 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, X 10 It is R, G, E, K, A, S, D, C, C, P, V and X 11is 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 Traf 2 / 3 sequence. 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, SEQ ID NO:42380.

[0035] In some embodiments, the protein comprises an amino acid sequence comprising the consensus C or XXXX, wherein: X is P, X is any amino acid, X is E, X is any amino acid, X is any amino acid, and X 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: X is Q, P, E, V, T, or S, X is I, L, M, N, S, T, N, D, and X is N, D, R, S, G, or Y; or the sequence is at least 60% identical to a). In some embodiments, the protein has the sequence PKQEXXXXX, SEQ ID NO: 42301. In some embodiments, the protein has the sequence XXXXPDDL, SEQ ID NO: 42381. In some embodiments, the protein has the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0036] In some embodiments, the invention relates to an amino acid sequence comprising: (a) an amino acid sequence of 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 at least 70% identical to the sequence in a); or (c) a sequence at least 80% similar to a).

[0037] In some embodiments, the TRAF domain of the CD40 protein comprises 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 at least 70% identical to the sequence in a); or (c) a sequence at least 80% similar to a).

[0038] In some embodiments is a TRAF6 domain sequence comprising: PQEINF, PEEMSW, PPENYE, or PQENSY.

[0039] In some embodiments is a TRAF2 / 3 domain sequence comprising: PVQET, TQEET, SKEET, AVEET, or PVQET.

[0040] In some embodiments is a TRAF2 domain sequence comprising: SVQE, AVEE, or PEEE.

[0041] In some embodiments, the TRAF 6 or TRAF2 / 3 domain comprises: (a) 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 at least 70% identical to a); or (c) an amino acid sequence at least 80% similar to a).

[0042] In some embodiments, the sequence is within the CD40 protein sequence of any of the proteins, amino acid sequences, or TRAF domains 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 domains within the CD40 protein.

[0043] In some embodiments, it is a TRAF2 / TRAF3 domain comprising 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, X9 is any amino acid, X 10 is any amino acid, and X 11is 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, SEQ ID NO:42378.

[0044] In some embodiments, it is a TRAF2 / TRAF3 domain comprising a 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, 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, SEQ ID NO:42380.

[0045] In some embodiments, the TRAF6 domain comprises a consensus amino acid sequence of C or XXXX, wherein XXX is P, X is any amino acid, X is E, X is any amino acid, X is any amino acid, and X is Ac / Ar. In some embodiments, the protein has the sequence PKQEX1X2X3X4X5X6, SEQ ID NO: 42301. In some embodiments, the protein has the sequence XXXX, SEQ ID NO: 42381. In some embodiments, the protein has the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0046] In some embodiments, the CD40 domain comprises one or more of the following amino acid sequences: (a) a TRAF2 / TRAF3 domain comprising 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, X9 is any amino acid, X 10 is any amino acid, and X 11 is any amino acid; (b) a TRAF2 / TRAF3 domain comprising a 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, X 10 is any amino acid, and X 11 is any amino acid; (c) a TRAF6 domain comprising a consensus amino acid sequence of C or 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.

[0047] In some embodiments, the chimeric co-stimulatory antigen receptor (CoStAR) comprises: an extracellular binding domain that binds to carcinoembryonic antigen (CEA) or an extracellular binding domain that binds to mesothelin (MSLN); a transmembrane domain connected 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 second signaling domain comprises a variant CD40 signaling domain or a signaling fragment thereof, wherein the variant CD40 comprises a sequence of a variant TRAF2 / TRAF3 sequence or a variant TRAF6 sequence or a variant TRAF2 sequence, 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 Figure 88A In some embodiments, the construct is as shown. Figure 88B In some embodiments, the construct is as shown. Figure 88C In some embodiments, the first signaling domain comprises a full-length costimulatory domain. In some embodiments, the extracellular binding domain is operably connected to the transmembrane domain via a linker and / or a 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 from CD28, CD8, ICOS, DAP10, or NTRK. 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 extracellular binding domain comprises an scFv, a peptide, an antibody heavy chain variable domain, an antibody light chain variable domain, or a CEA ligand.

[0048] In some embodiments is a nucleic acid encoding a CoStAR according to any of the embodiments of the present disclosure.

[0049] In some embodiments is a vector comprising a nucleic acid according to any embodiment of the present disclosure.

[0050] In some embodiments, the cells are cells expressing the CoStAR of any one embodiment of the present disclosure. In some embodiments, the cells include α-β T cells, γ-δ T cells, regulatory T cells, TILs, NKT cells, or NK cells. In some embodiments, the cells co-express CAR or TCR.

[0051] In some embodiments are methods of making a cell of any embodiment of the present disclosure comprising the step of transducing or transfecting the cell with a vector of any embodiment of the present disclosure.

[0052] In some embodiments are methods for preparing a cell population expressing a CoStAR according to any embodiment of the present disclosure, comprising: detecting expression of CoStAR on the surface of cells transfected or transduced with the vector of Alternative 62; and selecting cells identified as expressing said CoStAR.

[0053] In some embodiments are cell populations enriched for cells expressing the CoStAR of any embodiment of the disclosure.

[0054] In some embodiments are methods for treating a disease in a subject comprising the step of administering to the subject a cell of any embodiment of the disclosure or a population of cells of any embodiment of the disclosure.

[0055] 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 Figure 92 Any of the constructs shown (CTP188-CTP202).

[0056] Therefore, it is the intention of the present disclosure not to encompass any previously known product, method of making the product, or method of using the product within the present disclosure, so that the applicant reserves the right and hereby discloses a disclaimer of any previously known product, process, or method. It should also be noted that the present disclosure is not intended to encompass within the scope of the present disclosure any product, process, or method of making the product or using the product that does not meet the written description and implementation requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), so that the applicant reserves the right and therefore discloses a disclaimer of any previously described product, method of making the product, or method of using the product. In the practice of the present disclosure, it may be advantageous to comply with Art. 53(c) EPC and Rule 28(b) and (c) EPC. The right to expressly waive any embodiment that is the subject of any granted patent of the applicant in any previously filed application of the pedigree of this application or any other pedigree or any third party is expressly reserved. Nothing herein should be interpreted as a commitment.

[0057] It should be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” “comprising,” etc. may have the meanings ascribed to them in U.S. patent law; for example, they may mean “includes,” “included,” “including,” etc.; and terms such as “consisting essentially of / consists essentially of” have the meanings ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of any embodiment of the disclosure.

[0058] These and other embodiments are disclosed by or are obvious from and encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The following detailed description, which is given by way of example only and is not intended to limit any embodiment of the invention to the specific embodiments described, is best understood when read in conjunction with the accompanying drawings.

[0060] Figure 1 - Structural organization of single and fused costimulatory domain receptors. Schematic diagrams of the CoStAR receptors shown in the claims are shown. First, a CoStAR based on a single costimulatory receptor, followed by a fusion CoStAR consisting of a full-length costimulatory receptor signaling domain fused to a second costimulatory domain.

[0061] Figure 2Ai -2E- Genomic organization of potential CoStAR configurations - 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 from a promoter alone ( Figure 2Ai -2Aii), where CoStAR consists of a single ( Figure 2Ai ) or fusion ( Figure 2Aii )Co-stimulatory receptor composition; ( Figure 2B ) can be expressed with an epitope tag (e.g., His tag, DYKDDDDK (SEQ ID NO: 449), etc.) at the N-terminus or C-terminus to achieve direct staining of CoStAR; ( Figure 2C ) together with a marker gene separated using a 2A cleavage sequence or an internal ribosome entry site (IRES); ( Figure 2D ) together with a marker gene expressed by a second promoter; ( Figure 2E ) together with a protein of interest, such as a chimeric antigen receptor or T cell receptor, that is isolated using a 2A cleavage sequence or an internal ribosome entry site (IRES); ( Figure 2F ) together with a protein of interest, such as a chimeric antigen receptor or T cell receptor, expressed by a second promoter. For individuals with sufficient knowledge, it is clear that CoStAR and marker genes / chimeric antigen receptors / T cell receptors / other proteins of interest can be oriented to each other or expressed 3' or 5'.

[0062] Figure 3A Functional activity of CoStAR in 3E-T cells in response to antigens presented by LS174T and LoVo tumors. Figure 3A &3D), donor 2 ( Figure 3B ) and donor 3 ( Figure 3C Normal donor T cell populations of LS174 T cells (100 μg / mL) were lentivirally engineered to express CoStAR targeting carcinoembryonic antigen and magnetically sorted using CD34 magnetic selection to enrich for transgenes. T cells were mixed with wild-type unengineered CEA+ tumor cells (non-activated tumors) or CEA+ tumor cells engineered to express cell surface-anchored anti-CD3 single-chain antibody fragments (activated tumors) at the indicated effector to target ratios, and IL-2 in the supernatant was measured by ELISA. LS174 T cells (100 μg / mL) were used. Figure 3A , 3B&3C) and LoVo(3D&3E) obtained data.

[0063] Figure 4A -4D-CoStAR affects T cell proliferation. Figure 4A ) or does not exist ( Figure 4B)IL-2, 5×10 5 transduced and non-transduced T cells with 6.25×10 4 Wild-type LoVo or LoVo-OKT3 cells were mixed and the cells were counted three days later. In another assay at the same cell ratio, T cells from two donors ( Figure 4C and 4D) were loaded with proliferation dye, and the number of proliferation cycles undergone by the cells was determined by dye dilution using flow cytometry 6 days later.

[0064] Figure 5 -IL-2 activity of CoStAR fusion receptors in primary human T cells. Normal donor CD8+ T cells from 7 donors (except control CoStAR, which was 3 donors) were lentivirally transduced with the indicated CEA-targeted CoStAR and stimulated overnight in the presence of LoVo-OKT3 cells to assess IL-2 production. Intracellular flow staining was used to determine the proportion of IL-2 positive cells in CD34 negative (non-CoStAR transduction) and CD34+ (CoStAR transduction) populations. Asterisks show significant differences using paired Wilcoxon signed rank tests between transduced and non-transduced populations, *p<0.05.

[0065] Figures 6A-6D -Multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were lentivirally transduced with the indicated CEA-targeted CoStAR and assessed for IL-2 production after overnight stimulation in the presence of LoVo-OKT3 cells. IL-2 production was measured using intracellular flow staining in CD34-negative (non-CoStAR transduced) and CD34+ (CoStAR transduced) populations (7 donors) ( Figure 6A ), IFNγ (7 donors) ( Figure 6B )、BCL-xL(5 donors)( Figure 6C ) and CD107a (6 donors) ( Figure 6D ) The proportion of cells positive for the specific readout was quantified. The control was nonspecific CA125 targeting CoStAR and was derived from three donors in each case. The heat map is the average of all donors, with color intensity correlating to the percentage of cells positive for a specific readout under the defined conditions.

[0066] Figure 7-CD40 enhances IL-2 production from CD28-based CoStAR. Primary human T cells from three healthy donors were either kept untransduced or transduced with CoStARs based on truncated CD28 (TrCD28), full-length CD28 (FLCD28), or CD28.CD40 extracellular domains, with CEA-specific scFv (MFE23). Transduced cells were selected using the CD34 marker gene and expanded before analysis. Before IL-2 production was analyzed by ELISA, T cells were mixed with CEA+LoVo cells expressing OKT3 at an effector to target ratio of 8:1 for 20 hours.

[0067] Figure 8 Effects of signaling domains and target antigens on CoStAR-mediated T cell expansion. T cells were transduced with a CD28 or CD28.CD40-specific CoStAR or FolR-binding peptide (C7) based on the DYKDDDDK (SEQ ID NO: 449) epitope tag, harboring CA125, FolR, or CEA-specific scFvs. T cells were mixed with the OKT3-expressing CA125+ / FolR+ / CEA- cell line OVCAR3. The number of transduced cells was counted every 7 days until day 21, with fresh OVCAR3 cells added after each count.

[0068] Figure 9 Effects of signaling domains and target antigens on CoStAR-mediated T cell expansion. T cells were transduced with a CD28 or CD28.CD40-specific CoStAR or FolR-binding peptide (C7) based on the DYKDDDDK (SEQ ID NO: 449) epitope tag, harboring CA125, FolR, or CEA-specific scFvs. T cells were mixed with the OKT3-expressing CA125+ / FolR+ / CEA- cell line OVCAR3. The number of transduced cells was counted every 7 days until day 21, with fresh OVCAR3 cells added after each count.

[0069] Figures 10A-10B -( Figure 10A and 10B) CD40-based CoStAR enhances T cell costimulation in a TCR transfer model. Primary human T cells from three healthy donors were transduced with a CEA-specific TCR plus either a DYKDDDK-labeled CD28 or CD28.CD40-based CoStAR harboring either MFE (open or filled circles) or CA125 (open squares)-specific scFv. 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.

[0070] Figure 11 The enrichment and expansion of primary human T cells transduced to express the co-stimulatory molecules of the present invention are described. MFE23 is a single chain Fv antibody with high affinity for carcinoembryonic antigen (CEA). Primary human T cells were mock transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 CoStAR (each with a CD34 marker gene separated by a 2A cleavage peptide). After in vitro culture, MACS was used to TM Cells were enriched for CD34 using paramagnetic selection reagent (Miltenyi Biotech) and then expanded in number using irradiated feeder cells. An exemplary image from one of three donors is shown.

[0071] Figures 12A-12D Depicts the expansion of T cells transduced with co-stimulatory 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 co-cultured with LoVo-OKT3 cells at an effector: target ratio of 8:1 in the presence (200 IU / ml) or absence of exogenous IL-2. On days 1, 4, 7, 11, and 18, cells were harvested and the number of viable T cells was counted on a MACSQuant flow cytometer using anti-CD2 reagent. ( Figure 12A ) In the absence of tumor stimulation and IL-2, cell numbers decreased, as expected. ( Figure 12B ) In the absence of stimulation but in the presence of IL-2, there was more pronounced cell survival but no specific growth. ( Figure 12CIn the presence of tumor but without IL-2, mock cells showed no specific survival. MFE23.CD28 CoStAR-mediated expansion mediated a significant doubling within the first 4 days, followed by a decline. MFE23.CD28.CD40 mediated a greater expansion until day 7, followed by a steady decline. Figure 12D ) Under identical conditions but in the presence of IL-2, mock- and MFE23.CD28-transduced cells exhibited a 20-fold expansion over 18 days, whereas MFE23.CD28.CD40 cells expanded over 60-fold. Thus, CD28.CD40-based receptors exhibit superior expansion and survival in the presence and absence of exogenous IL-2 stimulation.

[0072] Figures 13A-13M Cytokine production by mock, MFE23.CD28, or MFE23.CD28.CD40 engineered T cells is depicted. Bead array analysis was performed on supernatants obtained from T cell / tumor co-cultures. 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 the same number of T cells alone or LoVo-OKT3 cells alone. TM Human TH1 / TH2 Cytokine Panel (Legendplex TM Human TH1 / TH2 cytokine panel (Biolegend) was used to analyze cytokine production. 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;( Figure 13M)IL-21. Cytokines were very low or not detected in culture media from individual T cells or tumors. When co-cultured with tumors, cytokine production was enhanced. Compared to the simulation, MFE23.CD28 enhanced the production of IL-2, IL-5, IL-17A / 17F, IL-10, IL-9, and IL-21. MFE23.CD28.CD40 also enhanced the production of TNFα, IL-13, and IL-22. MFE23.CD28.CD40 also enhanced the production of a large number of cytokines (IL-2, IL-9, and IL-17F) that are greater than the number of cytokines provided by MFE23.CD28, and reduced the production of some cytokines (IL-5 and IL-10) to below the levels seen with MFE23.CD28. These data together demonstrate that adding CD40 to CD28-based co-stimulatory receptors enhances and / or regulates their specific activity for chemokine production.

[0073] Figures 14A-14M Depicts the use of Legendplex TM Human proinflammatory chemokine panel (Legendplex TM HumanPro inflammatory chemokine panel) for analysis of chemokines. 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(CXCI1)( Figure 14L )I-TAC(CXCL11)( Figure 14M)MEP-1β(CCL4). Chemokines were very low or not detected in culture medium from T cells alone. When co-cultured with tumors, chemokine production was enhanced. Compared to the mock, MFE23.CD28 enhanced the production of CXCL5, CXCL10, CXCL11, CCL17, and CCL20. MFE23.CD28.CD40 also enhanced the production of CCL2, CXCL1, and CXCL9. MFE23.CD28.CD40 also enhanced the production of a large number of cytokines (CXCL1, CXCL9, CXCL10, CXCL11, CCL17, CCL2, CXCL9, CCL5, and CCL20), a greater number of cytokines than provided by MFE23.CD28, and reduced the production of some cytokines to below the levels seen with MFE23.CD28(CCL4). Together, these data demonstrate that the addition of CD40 to CD28-based costimulatory receptors enhances and / or modulates their specific activity towards chemokine production.

[0074] Figures 15A-15H Describe the functional activity of ovarian CoStAR engineered cells using CoStAR with FolR or CA125 reactive scFv (MOV19 & 196-14, respectively). Human folate receptor alpha (FolR) represents a suitable target for a variety of tumors (including ovarian, head and neck, kidney and lung), and CA125 represents an alternative target for ovarian cancer. Primary human T cells from 6 healthy donors were engineered with 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28 or MOV19.CD28.CD40 receptors, all of which have the DYKDDDDK epitope tag for detection. Before using intracellular staining to analyze effector activity in epitope tag positive and negative populations, transduced cells were mixed with FolR+ / CA125+OVCAR-OKT3 cells. In CD28 and CD28.CD40 engineered cells, responses to CA125 and FolR were observed through the production of IL-2 ( Figure 15A and 15B ), TNFα( Figure 15C and 15D )、CD137( Figure 15E and 15F ) and BCL-xL( Figure 15G and 15H ), except that no specific BCL-xL induction was observed by MOV19.CD28 compared to MOV19.CD28.CD40.

[0075] Figures 16A-16FDepicted are three TIL populations that were mock transduced or engineered with MOV19.CD28.CD40CoStAR and then mixed with patient-matched tumor digests. Donor tumors displayed varying levels of FolR on the digests, ranging from negative ( Figure 16A ), low expression ( Figure 16B ) to high expression ( Figure 16C CoStAR-negative TILs in mock- and FolR-negative digest-matched CoStAR-engineered TIL populations exhibited similar levels of CD137 upregulation following tumor co-culture, which was not enhanced by the presence of CoStAR ( Figure 16D In TILs exposed to digests low in FolR expression, activity was enhanced in CoStAR+ cells compared to CoStAR-, with CD137 expression increasing from <10% to >20% ( Figure 16E In TILs exposed to FolR-high tumor digests, activity increased from approximately 20% in the CoStAR- population to approximately 50% in the CoStAR+ population ( Figure 16F ).

[0076] Figures 17A-17C Enhancement of effector function is depicted. In response to FolR+ tumor digests, CoStAR targeting FolR enhanced CD137 expression from ∼20% to ∼50% ( Figure 17A ), increased TNFα production from 10% to 15% ( Figure 17B ) and enhanced IL-2 production from 2% to 5% ( Figure 17C ).

[0077] Figures 18A-18F The results 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 and activated with fixed OKT3 to provide stimulation in the absence of FolR, or with OvCAR-OKT3 to provide TCR and CoStAR activity. After OKT3 stimulation, BCL-xL activity increased by 10% to 20% in the three donors ( Figure 18A ), while IL-2 increased by 0% to 12% ( Figure 18B ), TNFα increased by 0% to 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 ∼20% in CD28 CoStAR but by ∼35% in CD28.CD40 CoStAR ( Figure 18D), IL-2 induction was enhanced by ∼20% in CD28 CoStAR but by 30–50% in CD28.CD40 CoStAR ( Figure 18E ), and TNFα production was enhanced by 20-30% in CD28 CoStAR and 25-50% in CD28.CD40 CoStAR ( Figure 18F Exogenous soluble FolR had no inhibitory effect on any of these effector functions.

[0078] Figure 19 Depicts surface expression of anti-MSLN CoStAR expression on HD T cells. Transduced and non-transduced (mock) cells underwent a rapid expansion protocol (REP) and the transduction efficiency of the cells was assessed using anti-CD34-APC (black) or anti-MSLN-PE antibodies (red) via surface detection of the marker gene tCD34 (truncated CD34) or CoStAR molecules. Results represent 3 biological replicates.

[0079] Figures 20A-20C Depicts cytokine expression in healthy donor (HD) T cells transduced with anti-MSLN CoStAR and co-cultured with OVCAR-3 cell lines. CoStARs comprising six different anti-MSLN scFvs (SS1, M5, HN1, M912, huYP218, P4) and three different spacer / transmembrane domains (CD28, N-terminally truncated CD28, CD8) were compared. Structural details are provided in Tables 8, 9, and 10. Shown are the cytokine expression profiles of healthy donor (HD) T cells co-cultured with OVCAR-3 or OVCAR3-OKT3 cell lines ( Figure 20A )IL-2, ( Figure 20B )IFNγ and ( Figure 20C ) TNFα cytokine concentration. Untreated T cells were used as controls. Results represent 1-3 biological replicates, each with 3 technical replicates.

[0080] Figures 21A-21C Depicts cytokine expression in healthy donor (HD) T cells transduced with anti-MSLN CoStARs and co-cultured with K562 cell lines. CoStARs containing six different anti-MSLN scFvs (SS1, M5, HN1, M912, huYP218, P4) and three different spacer / transmembrane domains (CD28, N-terminally truncated CD28, CD8) were compared. Structural details are provided in Tables 8, 9, and 10. Shown are the cytokine expression profiles of healthy donor (HD) T cells after co-culture with K562-MSNL or K562-MSNL-OKT3 cell lines ( Figure 21A )IL-2, ( Figure 21B )IFNγ and ( Figure 21C) TNFα cytokine concentration. Untreated T cells were used as controls. Results represent 1-3 biological replicates, each with 3 technical replicates.

[0081] Figure 22 Depicts surface expression of MFE23 scFV anti-CEA CoStAR expressed with six different secretion signal peptides (OSM1, CD8, CD2, IL2, GMCSF, hIgGκ). Structural details are provided in Table 11. After expansion, cell transduction efficiency was assessed via surface detection of marker gene-truncated CD34 (tCD34) or CoStAR molecules using anti-CD34-APC (black bars) or rhCEACAM5-Fc primary antibody and anti-IgG-Fc-PE secondary antibody (grey bars), respectively.

[0082] Figure 23 Surface expression of CoStAR containing six different anti-CEA scFvs (MFE23, MFE23 (Q>K), hMFE23, CEA6, BW431 / 26, hT84.66) is depicted. Structural details are provided in Table 12. After expansion, cell transduction efficiency was assessed via surface detection of markers, genetically truncated CD34 (tCD34), or CoStAR molecules, using anti-CD34-APC (black bars) or rhCEACAM5-Fc primary antibody and anti-IgG-Fc-PE secondary antibody (grey bars), respectively.

[0083] Figures 24A-24C 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 lines is depicted. Structural details are provided in Table 12. Cytokine production by healthy donor (HD) T cells after co-culture with Lovo or Lovo-OKT3 cell lines ( Figure 24A )IL-2, ( Figure 24B )IFNγ and ( Figure 24C ) TNFα cytokine concentration. Untreated T cells were used as negative control.

[0084] Figures 25A-25C Figure 3 depicts cytokine production by healthy donor (HD) T cells transduced with anti-CEA CoStARs (MFE23, MFE23(Q>K), hMFE23, CEA6, BW431 / 26, hT84.66) and co-cultured with K562 cell lines. Figure 3 shows cytokine production by healthy donor (HD) T cells co-cultured with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines. Figure 25A )IL-2, ( Figure 25B)IFNγ and ( Figure 25C ) TNFα cytokine concentration. Untreated T cells were used as negative control.

[0085] Figure 26 Depicts the surface expression of hMFE23 scFV anti-CEA CoStAR expressed with three different spacer / transmembrane domains. Structural details are provided in Table 13. After amplification, cell transduction efficiency was assessed by surface detection of marker gene-truncated CD34 (tCD34) using anti-CD34-APC (black bars) or by surface detection of CoStAR molecules using rhCEACAM5-Fc primary antibody and anti-IgG-Fc-PE secondary antibody (grey bars).

[0086] Figures 27A-27C Depicted are cytokine production by MFE23 scFV anti-CEA spacer variants. Shown are the cytokine production by MFE23 scFV anti-CEA spacer variants after co-culture with Lovo or Lovo.OKT3 cell lines ( Figure 27A )IL-2, ( Figure 27B )IFNγ and ( Figure 27C ) TNFα cytokine concentration. Untreated T cells were used as a control.

[0087] Figure 28 Depicted is an anti-CEA CoStAR comprising the hMFE23 CEA binding domain and an intracellular signaling domain containing CD40, CD134, CD137, CD2, ICOS, DAP10, and NTRK1 signaling elements.

[0088] Figure 29 Depicts the inclusion Figure 28 Surface expression of hMFE23 scFV anti-CEA CoStAR with domain combinations described in Figure 1 and detailed in Table 14. After expansion, cell transduction efficiency was assessed using anti-CD34-APC (black circles) via surface detection of marker gene-truncated CD34 (tCD34) or using rhCEACAM5-Fc primary antibody and anti-IgG-Fc-PE secondary antibody (red circles) via surface detection of CoStAR molecules. Results represent three biological replicates.

[0089] Figure 30 The T cell phenotype of CoStAR-transfected HD T cells in three separate donors is depicted. Cells were sorted using CD34 microbeads and subjected to a 14-day rapid expansion protocol (REP). After growth and REP, 1 × 10 5Differentiation subtypes of individual cells. Tcm, central memory T cell; Tem, effector memory T cell; Tn, naive T cell; Tscm, stem cell memory T cell; Tte, terminal effector T cell. T cell subtype definitions are shown in Table 15.

[0090] Figures 31A-31C Depicts cytokine production by hMFE23 scFV anti-CEA CoStAR transduced HD T cells co-cultured with K562 cell lines. Shown are the cytokine production by HD T cells co-cultured with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines. Figure 31A )IL-2, ( Figure 31B )IFNγ and ( Figure 31C ) TNFα cytokine concentration. Untreated T cells were used as a control.

[0091] Figures 32A-32C Depicts cytokine expression in HDT cells transduced with hMFE23 scFV anti-MSLN CoStAR and co-cultured with K562 cell lines. Cytokine expression after co-culture with K562.CEACAM5 (signal 2) or K562.CEACAM5.OKT3 (signal 1+2) cell lines is shown. Figure 32A )IL-2, ( Figure 32B )IFNγ and ( Figure 32C ) The frequency of cells expressing TNFα. Untreated T cells were used as a control.

[0092] Figure 33 Depicted is the proliferation of HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR in co-culture with K562.CEACAM5.OKT3 (Signal 1+2) cell lines. HD T cells were obtained from two donors. Graphs represent fold expansion of input cells.

[0093] Figure 34 Depicted is the proliferation of HD T cells transduced with hMFE23 scFV anti-MSLN CoStAR in co-culture with the K562.CEACAM5.OKT3 (Signal 1+2) cell line. HD T cells were obtained from two donors. The graph represents the fold expansion of the input cells on day 6 post-stimulation.

[0094] Figures 35A-35B TRAF and TRAF-like binding sites and motifs as well as signaling pathways are depicted. Figure 35A The CoStAR CD40 intracellular domain showing TRAF binding sites and signaling is depicted. Figure 35B The IProx domain-containing CoStAR is depicted.

[0095] Figures 36A-36B The effects of mutations in the intracellular signaling domain of CoStAR CD40 on cytokine secretion, long-term survival, and proliferation of CD28.CD40 CoStAR-transduced T cells co-cultured with LoVo.OKT3 are depicted. Cells from three donors were activated with Dynabeads and transduced with WT CD28.CD40 (CTP194), CD28.CD40 containing the TRAF2 binding site mutations SVQE>AVQA (CTP195), the TRAF2 / TRAF3 binding site mutations PVQET>AVAEA (CTP196), the TRAF6 binding site mutations PQEINF>AQAINF (CTP197), and Q263A (CTP199), or mock transduction. Figure 36A ) IL-2 was measured in supernatants collected 24 hours after co-culture with LoVo or LoVo.OKT3.GFP tumor cells in the absence of IL-2. ( Figure 36B Viability and absolute counts were assessed after 6-8 days, and viable T cells were re-stimulated with fresh LoVo.OKT3.GFP tumor cells for an additional week. At the end of long-term co-culture, viability and absolute counts were measured, and fold expansion was calculated. Data are shown as mean ± SEM for n ≤ 3 donors analyzed in triplicate.

[0096] Figures 37A-37B The percentage of TILs based on CD2+ staining in thawed OC samples on day 1 is depicted ( Figure 37A ) and total TIL counts ( Figure 37B ).

[0097] Figure 38 The growth of non-Td and Td TILs is depicted. Cell counts were performed using CD2 and DRAQ7 staining and acquired on a Novocyte 3005. Cell counts at the end of REP on day 25 are shown. Shapes corresponding to each of the five ovarian cancer samples are depicted. Filled shapes represent non-Td, and hollow shapes represent Td TILs. Statistical analysis was performed using a paired t-test.

[0098] Figures 39A-39D The transduction efficiency and viral integration per cell of CoStAR-modified TILs are depicted. On day 25 after REP, CD3+ ( Figure 39A )、CD4+( Figure 39B ) and CD8+( Figure 39C ) cell populations. Viral copy number (VCN) was assessed to determine viral integration ( Figure 39D). Shapes corresponding to each of the five ovarian cancer samples are depicted. Filled shapes are non-Td, and transparent shapes are TdTILs.

[0099] Figure 40 The CD4 and CD8 populations of TILs after REP are depicted. At D25 after REP, the CD4 and CD8 composition of non-transduced (non-Td), anti-FOLR1 CoStAR-transduced (Td), and anti-FOLR1 CoStAR+Td TILs were assessed by flow cytometry. Statistical analysis was performed using two-way ANOVA with matched Tukey's multiple comparison post-test.

[0100] Figures 41A-41C The effect of CoStAR modification on TIL differentiation status was depicted. At D25 after REP, total T cells ( Figure 41A )、CD4+( Figure 41B ) and CD8+( Figure 41C The differentiation status of non-transduced (non-Td), anti-FOLR1 CoStar transduced (Td) TILs, and anti-FOLR1 CoStar+Td TILs in the cell population was analyzed using two-way ANOVA and matched Tukey's multiple comparison test. Figure 41A ) and CD4+( Figure 41B ) population, statistically significant difference in Tscm population was observed between anti-FORL1 CoStAR-Td TIL and anti-FOLR1 CoStAR+Td TIL. *p<0.05

[0101] Figures 42A-42B The effects of CoStAR modification of TILs on the expression of co-inhibitory or co-stimulatory markers were characterized. Figure 42A ) and CD8+( Figure 42B ) Expression of co-inhibitory and co-stimulatory markers in non-transduced (non-Td), anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+Td TILs in the cell population. Shapes corresponding to each of the five ovarian cancer samples are depicted, regardless of fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR-Td, and anti-FOLR1 CoStAR+Td TILs, respectively. Statistical analysis was performed using two-way ANOVA with matched Tukey's multiple comparison post-test. *p < 0.05

[0102] Figures 43A-43BThe effects of CoStAR modification on TCRαβ, TCRγδ, and Treg frequencies in TILs are depicted. The frequencies of TCRαβ (CD3+TCRαβ+) and TCRγδ (CD3+TCRγδ+) in non-transduced (non-Td), anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+Td TILs were assessed in the CD3+ cell population. Figure 43A The frequency of Tregs in the CD4+ subset was also assessed ( Figure 43B ). Shapes corresponding to each of the five ovarian cancer samples are depicted without regard to fill. Black, dark gray, and light gray bars represent non-Td TILs, anti-FOLR1 CoStAR-Td TILs, and anti-FOLR1 CoStAR+Td TILs, respectively. Two-way ANOVA with matched Tukey's multiple comparison post-hoc test ( Figure 43A ) and one-way ANOVA with Friedman's post hoc test ( Figure 43B ) for statistical analysis.

[0103] Figures 44A-44C The effects of CoStAR modification of TILs on cytokine production following mitogenic activation were characterized. CD3+( Figure 44A )、CD4+( Figure 44B ) and CD8+( Figure 44C Figure 2. Cytokine production in non-transduced (non-Td), anti-FOLR1 CoStAR-transduced (Td) TILs, and anti-FOLR1 CoStAR+Td TILs after 4-hour stimulation with PMA (50 ng / mL) / ionomycin (1 μg / mL) in a 5% OC (OC) cell population. Shapes corresponding to each of the five OC samples are depicted, 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 a two-way ANOVA with a matched Tukey multiple comparison post hoc test. *p ≤ 0.05, ***p ≤ 0.001.

[0104] Figure 45 Depicted is the expression of FOLR1 on OC digested 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 the study are shown. Abbreviations: OC, ovarian cancer; FOLR1, folate receptor alpha; FOLR1 PE, anti-FOLR1 antibody conjugated to PE; PE, phycoerythrin.

[0105] Figures 46A-46DThe effect of CoStAR modification on cytokine-producing cells in co-culture with autologous tumors is depicted. Non-Td and anti-FOLR1 CoStAR Td TILs were co-cultured with autologous tumor digests for 16 hours, and intracellular flow cytometry was performed to assess the proportion of cytokine-producing cells. IL-2 positive ( Figure 46A )CD4 and ( Figure 46B )CD8 TIL frequency and TNFα positive ( Figure 46C )CD4 and ( Figure 46D ) CD8 TIL frequency. Results represent five biological replicates, each with three technical replicates. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test. *P < 0.05.

[0106] Figures 47A-47E The effect of CoStAR modification on cytokine secretion during co-culture with autologous tumor digests expressing FOLR1 is depicted. Non-Td and anti-FOLR1 CoStAR-transduced (Td) TILs were co-cultured with autologous tumor digests for 24 hours, and supernatants were collected and analyzed for cytokine secretion using MSD immunoassays. The graph shows the expression of ( Figure 47A )IL-2, ( Figure 47B )TNFα、( Figure 47C )IL-13 and ( Figure 47D )Measured concentration of IFNγ. ( Figure 47E )Correlation between IFNγ concentration and FOLR1 expression in autologous digests, e.g. Figure 1 Results are representative of five biological replicates, each with three technical replicates. Statistical analysis was performed using two-way ANOVA and Sidak's multiple comparison test. Correlations were analyzed using simple linear regression. *P < 0.05, **P < 0.01, ***P < 0.001.

[0107] Figure 48 Depicts the assessment of MHC-dependent CoStAR function in co-culture with autologous tumor digests. Non-Td and anti-FOLR1 CoStAR-transduced (Td) TILs were co-cultured with autologous tumor digests for 24 hours in the presence of MHC blocking reagents or isotype controls. The supernatants were then analyzed for IFNγ cytokine production using an MSD immunoassay. Results represent 3 biological replicates, each with 3 technical replicates. Statistical analysis was performed using a two-way ANOVA and Tukey's multiple comparison test.

[0108] Figures 49A-49BFigure 3. Effect of CoStAR modification on the frequency of cytokine-producing cells when co-cultured with engineered cell lines. Non-Td and anti-FORL1 CoStAR-transduced (Td) TILs were co-cultured with engineered target cell lines for 16 hours, and cytokine-producing cells were measured using intracellular flow cytometry. IL-2 positive frequencies (upper) and CD8+ (lower) TILs after co-culture with K-562 (left) and OVCAR-3 (right) derived lines. Figure 49A ). The frequency of TNFα positive in CD4+ (upper) and CD8+ (lower) TILs after co-culture with K-562 (left) and OVCAR-3 (right) derived lines ( Figure 49B Results are representative of five biological replicates, each with three technical replicates. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0109] Figures 50A-50D The effect of CoStAR modification on cytokine secretion during co-culture with engineered cell lines is depicted. Non-Td and anti-FOLR1 CoStAR-transduced (Td) TILs were co-cultured with engineered target cell lines for 24 hours, and MSD immunoassays were performed to assess secreted cytokine concentrations. Figure 3 shows the effect of CoStAR modification on cytokine secretion during co-culture with (left) K-562 and (right) BA / F3-derived cell lines. Figure 50A )IL-2, ( Figure 50B )TNFα、( Figure 50B )IL-13 and ( Figure 50D ) IFNγ cytokine concentration. Results are representative of five biological replicates, each with three technical replicates. 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.

[0110] Figures 51A-51C Evaluation of the effect of CoStAR modification on the cytotoxic capacity of TILs against OKT3-bearing targets is depicted. Non-Td and anti-FOLR1 CoStAR-transduced (Td) TILs were co-cultured with BA / F3 or OVCAR-3 engineered cell lines and analyzed by flow cytometry ( Figure 51A ) and xCELLigence assays ( Figure 51B and 51C )Evaluate cytotoxicity. Figure 51B )E:T ratio is 1:5 and ( Figure 51C) E:T ratio was 1:30. Results are representative of five biological replicates, each with three technical replicates. Flow cytometry data were statistically analyzed using two-way ANOVA with Tukey's multiple comparison test, and xCELLigence data were statistically analyzed using one-way ANOVA with Tukey's multiple comparison test. *P < 0.05, **P < 0.01.

[0111] Figures 52A-52D Depicts >80% viability ( Figure 52A ) and ~1-2 billion cells ( Figure 52B ), 40-55% of CD3+ T cells were CoStar transduced ( Figure 52C ) and >90% CD3+ T cell purity ( Figure 52D ) of NSCLC TILs.

[0112] Figure 53 Expansion of NSCLC TILs with >80% viability is depicted. There was no difference between CoStAR-transduced and non-transduced populations in terms of viability and cell expansion.

[0113] Figure 54 Figure 3: Expansion of NSCLC TILs with 0.5-5 billion cells at harvest. There were no differences in viability and cell expansion between CoStAR-transduced and non-transduced populations.

[0114] Figure 55 T cell populations of seven patient samples are profiled.

[0115] Figure 56 CD3 vs CD56 are depicted, where CD3+ marks T cells and CD3-CD56+ marks NK cells.

[0116] Figure 57 Quantification of non-transduced and CoStAR-transduced T cells is depicted.

[0117] Figure 58 CoStAR staining of T cell subsets is depicted.

[0118] Figures 59A-59D A significant increase in the viability of ovarian cancer TILs during REP was depicted, with viability >90% at the end of the preparation process, with 1-3 billion cells at harvest. Figure 59A Depicts the cell counts of ovarian cancer TILs over 10 days, and Figure 59B The viability of ovarian cancer TILs over 10 days is depicted. Figure 59C depicts the cell count after the preparation process, and Figure 59D Depicts the vibrancy after the preparation process.

[0119] Figures 60A-60D It is depicted that renal cancer TILs consistently displayed >50% viability during growth and >90% viability at the end of the production process, with 1.0-3.5 billion cells at harvest. Figure 60A depicts the cell counts of renal cancer TILs over 10 days, and Figure 60B The viability of renal cancer TILs over 10 days is depicted. Figure 60C depicts the cell count after the preparation process, and Figure 60D Depicts the vibrancy after the preparation process.

[0120] Figure 61 The experimental setup as outlined in Example 16 is depicted, in which CD40 signaling motif mutant constructs are incorporated into cells to screen for changes in transcription.

[0121] Figure 62A The experimental approach as outlined in Example 17 for the preparation of the TRAF variant CoStAR in T cells over 23 days is depicted.

[0122] Figure 62B The experimental approach as outlined in Example 17 for monitoring the effects of the TRAF variant CoStAR on T cell proliferation over 26 days is depicted.

[0123] Figures 63A-63C The total number of CD2+ T cells expressing the TRAF variant CoStAR is shown in Figure 2. Figure 63A )、HD 1004( Figure 63B ) and HD 2000( Figure 63C ) Graph showing changes in cell number of collected cells.

[0124] Figures 64A-64C Depicted are the fold changes in the number of CD2+ T cells expressing the TRAF variant CoStAR treated with IL-2. Figure 64A )、HD 1004( Figure 64B ) and HD 2000( Figure 64C ) Graph showing the fold change in cell number of collected cells.

[0125] Figures 65A-65C The total number of CD2+ T cells expressing the TRAF variant CoStAR is shown, where the T cells did not receive any IL-2 treatment. Figure 65A )、HD 1004( Figure 65B ) and HD 2000( Figure 65C ) Graph showing changes in cell number of collected cells.

[0126] Figures 66A-66CDepicted are the fold changes in the number of CD2+ T cells expressing the TRAF variant CoStAR, where the T cells did not receive any IL-2 treatment. Figure 66A )、HD 1004( Figure 66B ) and HD 2000( Figure 66C ) Graph showing the fold change in cell number of collected cells.

[0127] Figure 67 Some embodiments of CD40 TRAF variants are depicted as SEQ ID NOs: 510-519. In this figure, the underlined region is CD40, the sequence highlighted in grey is the TRAF6 binding domain region, the sequence highlighted in light grey is the TRAF2 / 3 binding domain, and the sequence highlighted in grey and enclosed in double brackets is the TRAF2 binding domain.

[0128] Figure 68 Depicted are the transduction levels of the constructs in primary T cells collected from donors HD 702C, HD 1004, and HD 2000.

[0129] Figures 69A-69F Describes the results of the experiment with IL-2 treatment ( Figures 69A-69C ) or without IL-2 treatment ( Figures 69D-69F ), the primary human donor 702C ( Figure 69A and 69D )、1004( Figure 69B and 69E ) and 2000( Figure 69C and 69F ) Fold change in cell proliferation of primary T cells expressing CoStAr constructs with TRAF6 variants compared to baseline.

[0130] Figures 70A-70F Describes the results of the experiment with IL-2 treatment ( Figures 70A-70C ) or without IL-2 treatment ( Figures 70D-70F ), the primary human donor 702C ( Figure 70A and 70D )、1004( Figure 70B and 70E ) and 2000( Figure 70C and 70F ) Fold change in cell proliferation compared to baseline of primary T cells expressing CoStAr constructs with TRAF2 / 3 variants.

[0131] Figures 71A-71F Describes the results of the experiment with IL-2 treatment ( Figures 71A-71C ) or without IL-2 treatment ( Figures 71D-71F ), the primary human donor 702C ( Figure 71A and 71D )、1004( Figure 71B and 71E ) and 2000( Figure 71C and 71F ) Fold change in cell proliferation compared to baseline of primary T cells expressing CoStAr constructs with TRAF2 variants.

[0132] Figures 72A-72F Describes the results of the experiment with IL-2 treatment ( Figures 72A-72C ) or without IL-2 treatment ( Figures 72D-72F ), the primary human donor 702C ( Figure 72A and 72D )、1004( Figure 72B and 72E ) and 2000( Figure 72C and 72F ) Fold change in cell proliferation of primary T cells expressing CoStAr constructs with different TRAF variants compared to baseline.

[0133] Figures 73A-73D Depicts Figures 69A-69C , 70A-70C, 71A-71C, and 72A-72C, where the fold change in cell proliferation compared to baseline for primary T cells from three donors is plotted over time. The graph shows cells treated with IL-2 and represents cells expressing TRAF6 ( Figure 73A )、TRAF2 / 3( Figure 73B )、TRAF2( Figure 73C ) or other TRAF( Figure 73D ) variants of the combined data of cells.

[0134] Figures 74A-74D Depicts Figures 69A-69C , 70A-70C, 71A-71C, and 72A-72C, which plot the fold change in cell proliferation compared to baseline for primary T cells from three donors over time. This figure shows cells that were not treated with IL-2 and represents cells expressing TRAF6 ( Figure 74A )、TRAF2 / 3( Figure 74B )、TRAF2( Figure 74C ) or other TRAF( Figure 74D ) variants of the combined data of cells.

[0135] Figures 75A-75B Depicts the IL-2 treatment at day 21. Figure 75A ) and without IL-2 treatment ( Figure 75B) compared to the combined fold change in cell proliferation of primary human T cells from three donors.

[0136] Figures 76A-76C A non-limiting example of a schematic model of a universal co-stimulatory protein with TRAF variants is depicted. Figure 76A TCR-incorporated antigen-agnostic receptors (TIAARs) include modified components of the TCR complex and associated signaling adaptors. Figure 76B ) A constitutive co-stimulatory receptor comprising a transmembrane domain (TMD) and characterized by the ability to be inducibly or constitutively activated. ( Figure 76C ) Inducible co-stimulatory receptors that 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*").

[0137] Figures 77A-77C Non-limiting exemplary protein constructs of some embodiments herein are depicted. Figure 77A is a general schematic diagram of a protein comprising the universal CoStAR sequence, further comprising optional parts, a binding domain, a CD28 domain, and a CD40 domain with TRAF variants. Figure 77B Schematic diagrams of some embodiments provided herein. Figure 77C A set of sequences of some embodiments provided herein is summarized.

[0138] Figure 78 A set of sequences (SEQ ID NOs: 40839-41074) of some embodiments provided herein are depicted. In some embodiments, these sequences are excluded from the consensus sequence or other TRAF variant constructs provided herein (including being excluded from Figure 84 and Figure 85 ).

[0139] Figure 79 A set of sequences (SEQ ID NOs: 41075-43474) of some embodiments provided herein are depicted. In some embodiments, these sequences are excluded from the consensus sequence or other TRAF variant constructs provided herein (including being excluded from Figure 84 and Figure 85 ).

[0140] Figure 80 Depicted is a non-limiting example of mutational analysis of CD40 using constructs CTP195, CTP196, and CTP197 as described in Example 13 to assess the function of the signaling motif.

[0141] Figure 81Depicted is a non-limiting example of mutational analysis of CD40 using constructs CTP195, CTP196, CTP197, CTP198, and CTP199 as described in Example 13 to assess the function of the signaling motif.

[0142] Figure 82A Depicted is the secondary consensus sequence generated from the alignment of variants in the TRAF2 domain shown in Table 1 , with highlighted amino acids conserved between variants.

[0143] Figure 82B The primary consensus sequence resulting from the alignment of variants in the TRAF2 domain shown in Table 1 is depicted, with highlighted amino acids conserved between variants.

[0144] Figure 82C Depicted is the consensus sequence generated by alignment of variants in the TRAF6 domain shown in Table 3, with highlighted amino acids conserved between variants.

[0145] Figure 83 Non-limiting example sequences of variants in the TRAF2 domain are depicted (SEQ ID NOs: 520-679).

[0146] Figure 84 Non-limiting example sequences of variants in the TRAF2 / 3 domain are depicted (SEQ ID NOs: 680-839).

[0147] Figure 85 Non-limiting example sequences of variants in the TRAF6 domain are depicted (SEQ ID NOs: 840-40839).

[0148] Figures 86A-86D Schematic diagrams showing some embodiments of FOLR1-αCoStAR and / or fusion proteins with CD40 variants are shown. Some general embodiments are depicted in Figure 86A middle. Figure 86B Some embodiments of FOLR1-αCoStAR or fusion proteins are depicted. Figure 86C Some embodiments of CoStAR or fusion proteins are depicted. Figure 86D Some embodiments of CoStAR or fusion proteins are depicted. In some embodiments, the sequence fully describes the structure and no further 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 can be used in the methods and / or constructs and / or proteins of the present invention, including Figure 28 、 67, 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0149] Figures 87A-87D Schematic diagrams showing some embodiments of anti-pembrolizumab CoStAR and / or fusion proteins with CD40 variants are shown. Some general embodiments are depicted in Figure 87A middle. Figure 87B Some embodiments of anti-pembrolizumab CoStAR or fusion proteins are depicted. Figure 87C Some embodiments of CoStAR or fusion proteins are depicted. Figure 87D Some embodiments of CoStAR or fusion proteins are depicted. In some embodiments, the sequence fully describes the structure and no further 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 can be used in the methods and / or constructs and / or proteins of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0150] Figures 88A-88D Schematic diagrams showing some embodiments of CEA CoStAR and / or fusion proteins with CD40 variants are shown. Some general embodiments are depicted in Figure 88A middle. Figure 88B Some embodiments of CEA CoStAR or fusion proteins are depicted. Figure 88C Some embodiments of CoStAR or fusion proteins are depicted. Figure 88DSome embodiments of CoStAR or fusion proteins are depicted. In some embodiments, the sequence fully describes the structure and no further 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 can be used in the methods and / or constructs and / or proteins of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (the Figure 78 and 79 excluding those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0151] Figures 89A-89D Schematic diagrams showing some embodiments of CEA CoStAR and / or fusion proteins with CD40 variants are shown. Some general embodiments are depicted in Figure 89A middle. Figure 89B Some embodiments of MSLN CoStAR or fusion proteins are depicted. Figure 89C Some embodiments of CoStAR or fusion proteins are depicted. Figure 89D Some embodiments of CoStAR or fusion proteins are depicted. In some embodiments, the sequence fully describes the structure and no further 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 can be used in the methods and / or constructs and / or proteins of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0152] Figures 90A-90C The cytokine TNF-α ( Figure 90A ), IFN-γ( Figure 90B ) and IL-2( Figure 90C ) amount.

[0153] Figures 91A-91BDepicted are IL-2 production in cells expressing one of the constructs CTP194-CTP200 as outlined in Example 20 ( Figure 91A ) and amplification factor ( Figure 91B ),.

[0154] Figure 92 Depicted is a schematic diagram of the protein construct CTP188-CTP202 as described herein. Detailed Description of the Invention

[0155] The disclosure herein relates to sequences encoding variants of TRAF binding domains. In some embodiments, these variants may be directed against TRAF2, TRAF2 / 3, or TRAF6 domains. In some embodiments, TRAF2 variants include one or more of the variants or point mutations provided herein. In some embodiments, TRAF2 / 3 variants include one or more of the variants or point mutations provided herein. In some embodiments, TRAF6 variants include one or more of the variants or point mutations provided herein. In some embodiments, TRAF2, TRAF2 / 3, or TRAF6 variants are any one or more of those provided 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, variants include those from Table 2, Table 4, Table 5, or Figure 67 In some embodiments, the variant is not Figure 78 (SEQ ID NO: 40839-41074) or Figure 79 (SEQ ID NO: 41075-43474). In some embodiments, the variant comprises Figure 83 、 Figure 84 or Figure 85 In some embodiments, the TRAF2, TRAF2 / 3, or TRAF6 variant is 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, such as Figures 76A-76C In some embodiments, the TRAF variant is part of a peptide construct, such as Figures 77A-77C , 86A-86D, 87A-87D, 88A-88D and / or 89A-89D.

[0156] In some embodiments, any of the proteins and / or CoStARs provided herein can use CD40 or TRAF variants in place of wild-type CD40 or TRAF domains. Any of the CD40, TRAF2, TRAF2 / 3, or TRAF6 variants provided herein can be used in the methods and / or constructs and / or proteins of the invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. For all descriptions of CD40, TRAF, TRAF2, TRAF2 / 3, and TRAF6 variants provided herein, in some embodiments, Figure 78 and 79 Sequences of are optionally excluded from the options of variants of the present invention.

[0157] 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 is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identical 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 is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identical 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 is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identical to any one of SEQ ID NOs: 840-40839. In some embodiments, the sequence comprises at least 2, 3, and / or 4 variants of TRAF.

[0158] In some embodiments, the sequence comprises a sequence from Table 2, 4, or 5. In some embodiments, the sequence comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identical 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 is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identical 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.

[0159] Some embodiments of the present disclosure relate to proteins comprising a protein comprising a 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, X 10 is any amino acid, and X 11 In some embodiments, X1 and X2 are any amino acids, 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-X 11 Any of X1-X2 is a naturally occurring amino acid. 11 In some embodiments, any one of X3 and / or X5 is a modified amino acid. In some embodiments, X3 and / or X5 are each conservatively mutated to another amino acid having similar properties to P and / or Q. 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 comprises: 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, X 10is 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 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, SEQ ID NO:42378.

[0160] In some embodiments, a protein is provided that comprises a consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of the sequence of 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, X 10 is any amino acid, and X 11 In some embodiments, X1 to X 11 Any of X1 to X2 is a naturally occurring amino acid. 11 Any one of X3 is a modified amino acid. In some embodiments, X3 is conservatively mutated to another amino acid with properties similar to P, S, A, and / or T. In some embodiments, X5 is conservatively mutated to another amino acid with properties similar to Q and / or E. In some embodiments, X6 is conservatively mutated to another amino acid with properties similar 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 comprises: 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, X 10It is 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 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, SEQ ID NO:42380.

[0161] In some embodiments, a protein is provided that comprises an amino acid sequence having a consensus C or XXXXXXX sequence, 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 to X6 is a naturally occurring amino acid. In some embodiments, any one of X1 to X6 is a modified amino acid. In some embodiments, X1 is conservatively mutated to another amino acid with similar properties 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 similar properties to acidic and / or aromatic amino acids. In some embodiments, X6 is selected from Y, H, W, or F amino acids. In some embodiments, X6 is selected from D or E amino acids. 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%, 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 has the sequence PKQEX1X2X3X4X5X6X7X8X9X 10 X11 , SEQ ID NO: 42301. In some embodiments, the protein has the sequence X1X2X3X4X5X6PDDL, SEQ ID NO: 42381. In some embodiments, the protein has the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0162] The consensus sequences A, B and C are shown in Tables 1 and 3 and Figures 82A-82C Assay shown.

[0163] In some embodiments, the amino acid sequence comprises: (a) one or more of SEQ ID NO: 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).

[0164] In some embodiments, the TRAF domain is in a CD40 protein, wherein the TRAF domain comprises 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; 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).

[0165] 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.

[0166] In some embodiments, the TRAF6 or TRAF2 / 3 domain comprises: (a) 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; 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).

[0167] In some embodiments, the sequence is within the CD40 protein sequence of the protein, amino acid sequence, or TRAF domain of any embodiment of the present disclosure. In some embodiments, the sequence is within the CD40 protein and is located at the corresponding TRAF2, TRAF2 / 3, and / or TRAF6 domain within the CD40 protein.

[0168] In some embodiments, the TRAF2 / TRAF3 domain comprises 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, X9 is any amino acid, 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, SEQ ID NO:42378.

[0169] In some embodiments, the TRAF2 / TRAF3 domain comprises 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, 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, SEQ ID NO:42380.

[0170] In some embodiments, the TRAF6 domain comprises an amino acid sequence of consensus C or XXXX, wherein XXX is P, X is any amino acid, X is E, X is any amino acid, X is any amino acid, and X is Ac / Ar. In some embodiments, the protein has the sequence PKQEX1X2X3X4X5X6, SEQ ID NO: 42301. In some embodiments, the protein has the sequence XXXX, SEQ ID NO: 42381. In some embodiments, the protein has the sequence PKQEX1X2X3X4X5X6PDDL, SEQ ID NO: 42382.

[0171] In some embodiments, the CD40 domain comprises one or more of the following amino acid sequences: (a) a TRAF2 / TRAF3 domain comprising 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, X9 is any amino acid, X 10 is any amino acid, and X 11 is any amino acid; (b) a TRAF2 / TRAF3 domain comprising a 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, X 10 is any amino acid, and X 11 is any amino acid; (c) a TRAF6 domain comprising a consensus amino acid sequence of C or 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.

[0172] In some embodiments, a chimeric co-stimulatory antigen receptor (CoStAR) is provided, comprising: an extracellular binding domain that binds to a tumor-associated antigen; a transmembrane domain connected 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 sequence of a variant TRAF2 / TRAF3 sequence or a variant TRAF6 sequence or a variant TRAF2 sequence, 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0173] 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 connected 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 sequence of a variant TRAF2 / TRAF3 sequence, or a variant TRAF6 sequence, or a variant TRAF2 sequence, 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 as Figure 88A In some embodiments, the construct is as shown. Figure 88B In some embodiments, the construct is as shown. 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0174] In some embodiments, the extracellular binding domain is operably connected to the transmembrane domain via a linker and / or a 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 number of amino acids between 2 and 350. 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.

[0175] In some embodiments, the transmembrane domain comprises a transmembrane domain from CD28, CD8, ICOS, DAP10, or NTRK. In some embodiments, the transmembrane domain comprises a transmembrane domain from CD28. In some embodiments, the transmembrane domain comprises a transmembrane domain from CD8. In some embodiments, the transmembrane domain comprises a transmembrane domain from ICOS. In some embodiments, the transmembrane domain comprises a transmembrane domain from DAP10. In some embodiments, the transmembrane domain comprises a transmembrane domain from 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 transmembrane domain comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer between about 60% and about 100% identical to any of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the extracellular binding domain comprises a 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 of the invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0176] In some embodiments is a nucleic acid encoding a CD40 TRAF variant construct according to any embodiment of the present disclosure.

[0177] In some embodiments is a nucleic acid encoding a CoStAR according to any of the embodiments of the present disclosure.

[0178] In some embodiments is a vector comprising a nucleic acid according to any embodiment of the present disclosure.

[0179] In some embodiments, the cells are cells expressing a CD40 TRAF variant construct of any embodiment of the present disclosure. In some embodiments, the cells include α-β T cells, γ-δ T cells, regulatory T cells, TILs, NKT cells, or NK cells. In some embodiments, the cells co-express a CAR or TCR.

[0180] In some embodiments, the cells are cells expressing the CoStAR of any one embodiment of the present disclosure. In some embodiments, the cells include α-β T cells, γ-δ T cells, regulatory T cells, TILs, NKT cells, or NK cells. In some embodiments, the cells co-express CAR or TCR.

[0181] In some embodiments are methods of making a cell of any embodiment of the present disclosure comprising the step of transducing or transfecting the cell with a vector of any embodiment of the present disclosure.

[0182] In some embodiments are methods for preparing a cell population expressing a CoStAR according to any embodiment of the present disclosure, comprising: detecting expression of CoStAR on the surface of cells transfected or transduced with the vector of Alternative 62; and selecting cells identified as expressing said CoStAR.

[0183] In some embodiments are cell populations enriched for cellular expression of a CoStAR according to any one of the embodiments of the present disclosure.

[0184] In some embodiments are methods for treating a disease in a subject comprising the step of administering to the subject a cell of any embodiment of the disclosure or a population of cells of any embodiment of the disclosure.

[0185] In some embodiments, the protein comprises an amino acid sequence of one of SEQ ID NOs: 510-519. In some embodiments, the protein comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical to any 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 that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical 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 that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical to SEQ ID NO: 511. In some embodiments, the protein comprises the sequence of SEQ ID NO: 512. In some embodiments, the protein comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical to SEQ ID NO: 512. In some embodiments, the protein comprises the sequence of SEQ ID NO: 513. In some embodiments, the protein comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical to SEQ ID NO: 513. In some embodiments, the protein comprises the sequence of SEQ ID NO: 514. In some embodiments, the protein comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical to SEQ ID NO: 514. In some embodiments, the protein comprises the sequence of SEQ ID NO: 515. In some embodiments, the protein comprises a sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical 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 that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical 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 that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical 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 that is about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100%, or any integer from about 60% to about 100% identical 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 from about 60% to about 100% identity to SEQ ID NO: 519. 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 of the invention, including. Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0186] In some embodiments, an immune cell comprising an extracellular tumor-associated antigen (TAA)-specific binding domain and a transmembrane domain comprises: a) a CD28 intracellular signaling domain; and b) a CD40 intracellular signaling domain, wherein the CD40 intracellular signaling domain comprises a TRAF variant according to any one embodiment of the present disclosure. In some embodiments, 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, wherein the nucleic acid molecule further comprises a TRAF variant according to any one embodiment 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 of the present invention, including Figure 28 、 67, 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0187] In some embodiments 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 according to any one embodiment 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 native antigen-binding polypeptide, such as but not limited to transferrin; or (e) a domain that binds to an antibody (e.g., an Fc binding domain, such as but not limited to CD16, CD32, or CD64), or other indirect methods of antigen recognition.In some embodiments, CoStAr is fused to at least one of: (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% and 100% sequence identity at the protein level, (2) an intracellular domain from human CD137 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, (3) an intracellular domain from human CD134 or (4) an intracellular domain from human CD2 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, (5) an intracellular domain 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 from human GITR or a variant thereof having a sequence identity of at least 60%, 70%, 80%, 90%, 95%, 99%, 100% or any integer from 60% to 100% at the protein level, (7) an intracellular domain from human IL2R-γ or a variant thereof having a sequence identity of at least 60%, 70%, 80%, 90%, 95%, 99%, 100% or any integer from 60% to 100% at the protein level, (8) an intracellular domain from human CD40 or a variant thereof having a sequence identity of at least 60%, 70%, 80%, 90%, 95%, 99%, 100% or any integer from 60% to 100% at the protein level, (i) an intracellular domain of a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100% or any integer from 60% to 100% sequence identity at the protein level, (ii) an intracellular domain of a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100% or any integer from 60% to 100% sequence identity at the protein level, and / or (iii) an intracellular domain of a variant thereof having at least 60%, 70%, 80%, 90%, 95%, 99%, 100% or any integer from 60% to 100% sequence identity at the protein level. 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 of the present invention, including. Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0188] 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 the at least one extracellular binding domain comprises the amino acid sequence of SEQ ID NO: 42275, wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 42276, and wherein 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 according to 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; a second portion comprising the transmembrane domain; a third portion comprising the amino acid sequence of SEQ ID NO: 42276; and a fourth portion comprising the amino acid sequence of SEQ ID NO: 42277, wherein the first portion is linked to the second portion, the second portion is linked to the third portion, and the third portion is linked to the fourth portion. In some embodiments, the protein further comprises at least one TRAF variant of any one embodiment of the present disclosure. In some embodiments is a protein comprising: 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 via the fifth portion, wherein the third portion is linked to the second portion, and wherein the fourth portion is linked to the third portion. In some embodiments, the protein further comprises at least one TRAF variant of any one embodiment 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 of the present invention, including Figure 28 、 67, 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0189] In some embodiments 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 the extracellular binding domain comprises the VH of antibody 196-14 and the VL of antibody 196-14, wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 42276, and wherein 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 according to any one embodiment of the present disclosure. In some embodiments, the CoStAR is a cell population enriched for cellular expression of a CoStAR comprising: an extracellular binding domain operably linked to a transmembrane domain, a CD28 signaling domain, and a CD40 signaling domain, wherein the extracellular binding domain comprises the VH of antibody 196-14 and the VL of antibody 196-14; wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 42276, and wherein 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 embodiment 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79The sequence options in can be optionally excluded from all embodiments provided herein.

[0190] 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 according to 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 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, CD155PD-1, mesothelin, or tumor-specific peptide (p)-major histocompatibility (MHC) complex, or 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0191] In some embodiments, it is a chimeric co-stimulatory 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 embodiment 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, 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, CD155PD-1, mesothelin, or tumor-specific peptide (p)-major histocompatibility (MHC) complex, or 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0192] In some embodiments is a fusion protein comprising: at least one TRAF variant according to any one embodiment of the present disclosure, (a) a binding domain, wherein the binding domain comprises: 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, a fusion protein comprises the amino acid sequence of SEQ ID NO: 42297 or 42298 and at least one TRAF variant of any one embodiment 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0193] In some embodiments, an engineered protein is provided comprising an aggregation domain and a signaling domain comprising a CD40 signaling domain or a signaling fragment thereof and at least one TRAF variant of any one embodiment of the present disclosure, wherein the aggregation domain is capable of oligomerization, thereby activating the signaling domain. In some embodiments, the protein comprises a constitutively stimulatory antigen-agnostic receptor (c-SAAR). In some embodiments, the protein comprises at least one of the following: LZ(cFos)-EGFR™ / JMD-CD28-CD40, LZ(cFos)-CD28™-CD28-CD40, LZ(cJun)-EGFR™ / JMDCD28-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(A30514-pembro)-CD28TMD-CD28-CD40, or anti-ID1-VL-VH(A30514-pembro)CD28TMD-CD28-CD40, or anti-ID2-VH-VL(A30523-pembro)-CD28TMD-CD28-CD40, or anti-ID2-VL-Vh(A30523-pembro)-CD28TMD-CD28-CD40, or anti-ID3-Vh-VL(A30633-pembro)-CD28TMD-CD28-CD40, or anti-ID3-VL-VH(A30633-pembro)-CD28TMD-CD28-CD40. In some embodiments, the engineered protein comprises a transmembrane domain, and when the engineered protein is bound to a ligand, the aggregation domain oligomerizes. 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79The sequence options in can be optionally excluded from all embodiments provided herein.

[0194] In some embodiments, it is a CoStAR comprising an extracellular binding domain that binds to carcinoembryonic antigen (CEA) and at least one TRAF variant of any one embodiment 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: HCDR1, which is the HCDR1 in SEQ ID NO: 12; HCDR2, which is the HCDR2 in SEQ ID NO: 12; HCDR3, which is the HCDR3 in SEQ ID NO: 12; LCDR1, which is the LCDR1 in SEQ ID NO: 12; LCDR2, which is the LCDR2 in SEQ ID NO: 12; and LCDR3, which is the HCDR3 in SEQ ID NO: 12. In some embodiments, a fusion protein comprising at least one TRAF variant of at least one embodiment of the present disclosure, wherein the fusion protein further comprises a HCDR1 that is the HCDR1 in SEQ ID NO: 12; a HCDR2 that is the HCDR2 in SEQ ID NO: 12; a HCDR3 that is the HCDR3 in SEQ ID NO: 12; a LCDR1 that is the LCDR1 in SEQ ID NO: 12; a LCDR2 that is the LCDR2 in SEQ ID NO: 12; and a LCDR3 that is the HCDR3 in SEQ ID NO: 12. In some embodiments, a construct such as Figure 88A In some embodiments, the construct is as shown. Figure 88B In some embodiments, the construct is as shown. 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0195] In some embodiments is a CoStAR comprising: an extracellular binding domain that binds to mesothelin (MSLN) and at least one TRAF variant according to any one embodiment of the disclosure. In some embodiments, the CoStAR further comprises a transmembrane domain; a CD28 signaling domain, wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 25; and a CD40 signaling domain, wherein the extracellular binding domain comprises any of the following: a HCDR1 which is the HCDR1 of SEQ ID NO: 186, a HCDR2 which is the HCDR2 of SEQ ID NO: 186, a HCDR3 which is the HCDR3 of SEQ ID NO: 186, a LCDR1 which is the LCDR1 of SEQ ID NO: 186, a LCDR2 which is the LCDR2 of SEQ ID NO: 186, and a LCDR3 which is the LCDR3 of SEQ ID NO: 186; a HCDR1 which is the HCDR1 of SEQ ID NO: 187, a HCDR2 which is the HCDR2 of SEQ ID NO: 187, a HCDR3 which is the HCDR3 of SEQ ID NO: 187, a LCDR1 which is the LCDR1 of SEQ ID NO: 187 : LCDR1, LCDR2, which are LCDR2 and LCDR3, which are LCDR3, of SEQ ID NO: 187; HCDR1, which are HCDR1, HCDR2, which are HCDR2 and HCDR3, which are HCDR3, of SEQ ID NO: 188; LCDR1, which are LCDR1, LCDR2, which are LCDR2 and LCDR3, which are LCDR3, of SEQ ID NO: 188; HCDR1, which are HCDR1, HCDR2, which are HCDR2 and HCDR3, of SEQ ID NO: 189; LCDR1, which are LCDR1 and LCDR2, which are SEQ ID NO: 189; LCDR2 and LCDR3 in NO: 189, which is LCDR3 in SEQ ID NO: 189;HCDR1 which is the HCDR1 of SEQ ID NO: 190, HCDR2 which is the HCDR2 of SEQ ID NO: 190, HCDR3 which is the HCDR3 of SEQ ID NO: 190, LCDR1 which is the LCDR1 of SEQ ID NO: 190, LCDR2 which is the LCDR2 of SEQ ID NO: 190, and LCDR3 which is the LCDR3 of SEQ ID NO: 190; HCDR1 which is the HCDR1 of SEQ ID NO: 191, HCDR2 which is the HCDR2 of SEQ ID NO: 191, HCDR3 which is the HCDR3 of SEQ ID NO: 191, LCDR1 which is the LCDR1 of SEQ ID NO: 191, LCDR2 which is the LCDR2 of SEQ ID NO: 191, and LCDR3 which is the LCDR3 of SEQ ID NO: 191; VH which is the VH of SEQ ID NO: 186, and VL which is the 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, a fusion protein comprises at least one TRAF variant of any one embodiment of the present disclosure. In some embodiments, the fusion protein further comprises a first sequence comprising any one or more of the following: 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;188; HCDR1 which is the HCDR1 of SEQ ID NO: 187, HCDR2 which is the HCDR2 of SEQ ID NO: 187, HCDR3 which is the HCDR3 of SEQ ID NO: 187, LCDR1 which is the LCDR1 of SEQ ID NO: 187, LCDR2 which is the LCDR2 of SEQ ID NO: 187, and LCDR3 which is the LCDR3 of SEQ ID NO: 187; HCDR1 which is the HCDR1 of SEQ ID NO: 188, HCDR2 which is the HCDR2 of SEQ ID NO: 188, HCDR3 which is the HCDR3 of SEQ ID NO: 188, LCDR1 which is the LCDR1 of SEQ ID NO: 188, LCDR2 which is the LCDR2 of SEQ ID NO: 188, and LCDR3 which is the LCDR3 of SEQ ID NO: 188; HCDR1 which is the HCDR1 of SEQ ID NO: 189, HCDR2 which is the LCDR2 of SEQ ID NO: 189 : 190; HCDR1 which is the HCDR1 of SEQ ID NO: 191, HCDR2 which is the HCDR2 of SEQ ID NO: 190, HCDR3 which is the HCDR3 of SEQ ID NO: 190, LCDR1 which is the LCDR1 of SEQ ID NO: 190, LCDR2 which is the LCDR2 of SEQ ID NO: 190, and LCDR3 which is the LCDR3 of SEQ ID NO: 190; HCDR1 which is the HCDR1 of SEQ ID NO: 191, HCDR2 which is the HCDR2 of SEQ ID NO: 191, HCDR3 which is the LCDR3 of SEQ ID NO: 190. HCDR3 of NO:191, LCDR1 which is LCDR1 of SEQ ID NO:191, LCDR2 which is LCDR2 of SEQ ID NO:191, and LCDR3 which is LCDR3 of SEQ ID NO:191; VH which is VH of SEQ ID NO:186, and VL which is VL of SEQ ID NO:186; VH which is VH of SEQ ID NO:187, and VL which is VI 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 as a transmembrane domain, a third sequence comprising the amino acid sequence of SEQ ID NO: 25; and a fourth sequence comprising the amino acid sequence of SEQ ID NO: 32, wherein the first sequence is linked to the second sequence, wherein the second sequence is linked to the third sequence, and wherein the third sequence is linked to the fourth sequence. 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 of the present invention, including: Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0196] In some embodiments, it is CoStAR, which comprises at least one TRAF variant of any one embodiment of the present disclosure, wherein when the T cell receptor (TCR) is engaged with the target of the TCR, CoStAR is able to perform CoStAR-ligand inducible activation on the immune cell carrying the TCR, comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain that are specific for the inducible ligand. In some embodiments, it is a cell expressing CoStAR, an antibody, or a protein of any one embodiment of the present disclosure. In some embodiments, the cell comprises α-β T cells. In some embodiments, the α-β T cells comprise tumor infiltrating lymphocytes (TIL). In some embodiments, CoStAR does not comprise a full-length CD28 extracellular domain. In some embodiments, 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 a signaling fragment thereof. In some embodiments, the ligand binding domain binds tafasitamab, cetuximab, trastuzumab, and / or pembrolizumab. 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 of the invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0197] In some embodiments is a method of cell therapy comprising identifying a subject in need thereof, and administering the CoStAR, fusion protein, nucleic acid, vector, or cell of any one embodiment of the disclosure.

[0198] In some embodiments are methods for preparing a cell population enriched for CoStAR or protein expressing a TRAF variant, the method comprising detecting expression of CoStAR or protein on the surface of cells transfected or transduced with a vector of any embodiment of the present disclosure, and selecting cells identified as expressing the CoStAR or protein.

[0199] In some embodiments are methods for treating a disease in a subject in need thereof, the method comprising administering to the subject a cell or cell population of any one embodiment of the present disclosure. CD40 and TRAF

[0200] 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. CD40 initiates signaling through multimerization of its ligands by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. The TRAF binding site is responsible for CD40's intracellular signaling.

[0201] The TRAF domain mediates the oligomerization of TRAF proteins and their association with upstream receptors or adaptors and downstream effector proteins. The RING domain is well known for its function in mediating protein ubiquitination within the large family of E3 ubiquitin ligases. Major downstream pathways regulated by TRAFs include those leading to activation of nuclear factor κβ (NF-κB) and mitogen-activated protein kinases (MAPKs), which in turn are important for the induction of genes related to innate immunity, inflammation, and cell survival. TRAF2 and TRAF3 function as negative regulators in several signaling pathways involved in B cell survival and inflammatory responses in innate immune cells.

[0202] The classical pathway of NF-κB is induced by TNFα, IL-1, or LPS and uses a variety of signaling adaptors to engage IKK activity. Phosphorylation of serine residues in the signal response region (SRR) of classical IκB by IKKβ leads to IκB ubiquitination and subsequent proteasomal degradation. This results in the release of NF-κB dimers, which can then translocate to the nucleus and induce transcription of target genes. The non-classical pathway relies on NIK (NF-κB inducing kinase)-induced IKKα activation. IKKα phosphorylates the p100 NF-κB subunit, which leads to the proteasomal processing of p100 to p52. This leads to the activation of the p52-RelB dimer, which targets specific κB elements.

[0203] It has been found that co-stimulatory receptors comprising a CD40 signaling domain show new and improved activity characteristics. Variants of the TRAF2, TRAF2 / 3, and TRAF6 binding domains within CD40 have also been found to regulate signal transduction. Activity characteristics can be regulated by selecting the intracellular domain of the receptor protein to connect to the CD40 signaling domain and / or by selecting elements of the CD40 signaling domain to connect to the intracellular domain of the receptor protein. Provided herein is a recombinant co-stimulatory antigen receptor (CoStAR) comprising: (i) a disease or tumor-associated antigen binding domain, (ii) a first intracellular segment containing an intracellular signaling domain of a receptor protein, and (iii) a second intracellular signaling domain of a CD40 receptor protein or a signal transduction fragment thereof. Optionally, CoStAR comprises 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 operates as a spacer of adjustable length, allowing the disease or tumor associated antigen binding domain to be moved away from the surface of the cell where it is expressed, for example to form a better immune synapse. In some embodiments, the extracellular segment and the first intracellular segment of the stimulatory receptor protein 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 an intervening transmembrane domain between the disease or tumor antigen binding domain and the first intracellular domain. When the extracellular segment of the stimulatory receptor protein is present, the transmembrane domain is 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 of the present invention, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC. Figure 78 and 79 The sequence options in can be optionally excluded from all embodiments provided herein.

[0204] As used herein, "full-length protein" or "full-length receptor" refers to a receptor protein such as, for example, a CD28 receptor protein. The term "full-length" encompasses receptor proteins that lack up to about 5 or up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus of the mature receptor protein once its signal peptide is cleaved. For example, while a specific cleavage site for the receptor N-terminal signal peptide can be determined, variability in the exact point of cleavage has been observed. The term "full-length" does not imply the presence or absence of amino acids of the receptor N-terminal signal peptide. In one embodiment, the term "full-length" (e.g., according to certain aspects of the invention, full-length CD28 or full-length CD40 intracellular domain) encompasses mature receptor proteins (e.g., according to certain aspects of the invention, CD28) that lack up to about 5, such as 1, 2, 3, 4, 5, or up to 10 amino acids, at the N-terminus of the mature receptor protein once its signal peptide is cleaved. As mentioned above, the "full-length" CD28 receptor or other receptor or tumor antigen binding domain according to various aspects of the present 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 the 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 area).

[0205] CoStARs have a modular format and can be constructed to contain extracellular, transmembrane, and intracellular domains obtained from one or more proteins, and a scFv obtained from an antibody that binds to a disease-associated antigen (eg, a tumor-associated antigen).

[0206] According to the present invention, in one embodiment, CoStAR includes disease-related (e.g., tumor-related) antigen receptors, such as, but not limited to, tumor-associated antigen-specific scFv, and can be combined with its cognate ligand and provide a primary co-stimulatory receptor protein for intracellular signaling. In some embodiments, primary co-stimulatory receptors can be smaller than full-length protein but sufficient to bind cognate ligands and transduce signals. In some embodiments, primary co-stimulatory receptor domains are full-length, such as, but not limited to, full-length CD28. Therefore, antigen-specific binding domains and ligand-specific receptors can each bind to cognate antigens and ligands. The amino acid sequence provided herein provides embodiments of several CoStAR constructs. These include CoStAR constructs comprising an antigen binding domain, an optional spacer, an optional co-stimulatory receptor protein, and an intracellular CD40 signaling domain, wherein the optional co-stimulatory receptor protein comprises an extracellular ligand binding segment or a fragment thereof. In another embodiment, CoStAR includes an antigen binding domain, an optional spacer, an extracellular ligand binding portion of a costimulatory receptor protein, a transmembrane domain, and a selected costimulatory receptor protein and an intracellular signaling domain of an intracellular CD40 signaling domain. In some embodiments, the extracellular ligand binding portion includes CD28 truncation, such as C-terminal CD28 truncation after amino acid 1EV, and is followed by an intracellular signaling domain. In some embodiments, the intracellular signaling domain is from CD40. The transmembrane domain separating the extracellular ligand binding and intracellular signaling domain can be from, but is not limited to, CD28, CD40. In a further embodiment, CoStAR can include additional costimulatory domains, such as the third intracellular costimulatory signaling domain, and in this respect can be similar to certain chimeric antigen receptors (CARs), which have been classified as first generation (only CD3 ζ), second generation (one costimulatory domain+CD3 ζ) or third generation (more than one costimulatory domain+CD3 ζ).

[0207] The co-stimulatory receptor proteins that can be used for 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, the native form of which comprises an extracellular ligand binding domain and an intracellular signal transduction domain. For example, CD2 is characterized as a cell adhesion molecule found on the surface of T cells and is capable of initiating the intracellular signal necessary for T cell activation. CD27 is characterized as a type II transmembrane glycoprotein belonging to the TNFR superfamily (TNFRSF), and its expression on B cells is induced by antigen receptor activation in B cells. CD28 is one of the proteins on T cells and is a receptor for CD80 (B7.1) and CD86 (B7.2) ligands on antigen presenting cells. CD137 (4-1BB) ligands are found on most leukocytes and some non-immune cells. OX40 ligands are expressed on many antigen presenting cells such as DC2s (dendritic cells), macrophages and B lymphocytes. In one embodiment, the co-stimulatory receptor protein is full-length CD28 as defined herein.

[0208] CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and produces several isoforms through alternative splicing. Its ligand, CD154 (also known as CD40L), is a protein expressed primarily on activated T cells. For reference, the human CD40 isoform 1 protein sequence is shown in GenBank Accession No. NP_001241.1, including 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, including amino acid sequences that conform to the SH3 motif (KPTNKAPH) (SEQ ID NO: 35), the TRAF2 motif (PKQE (SEQ ID NO: 36), PVQE (SEQ ID NO: 37), SVQE (SEQ ID NO: 38)), the TRAF6 motif (QEPQEINFP) (SEQ ID NO: 39), and the PKA motif (KKPTNKA (SEQ ID NO: 40), SRISVQE (SEQ ID NO: 41)). The present invention also includes engineered signaling domains comprising TRAF-binding amino acid sequences, such as engineered CD40 signaling domains.The engineered signaling domain that binds to TRAF1, TRAF2, TRAF3, and TRAF5 can comprise a major consensus sequence (P / S / A / T)X(Q / E)E or a minor consensus sequence PXQXXD and can be identified in or obtained from, but not limited to, TNFR family members such as 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 Sequences). 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).

[0209] The embodiments disclosed herein demonstrate the operation of CD40 as a co-stimulatory signaling domain in CoStAR, and further demonstrate that cytokine and chemokine expression characteristics are changed by signaling domain selection. In some embodiments, the co-stimulatory CD40 signaling domain of CoStAR promotes proinflammatory cytokines (e.g., IL-2, TNFα). In some embodiments, the co-stimulatory CD40 signaling domain of CoStAR reduces immunosuppressive cytokines (e.g., IL-5, IL-10). The co-stimulatory activity of the CD40 signaling domain or fragment can 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, as compared to the activity of the first receptor signaling domain without the CD40 signaling domain or fragment.In this regard, the CD40 signaling domain of the invention (including signaling fragments comprising a specific factor binding site or in which the specific factor binding site is mutated) combined with the costimulatory first signaling domain can promote or inhibit 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, e.g., 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 L et al., TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages. J Immunol. 2003; 33: 779-789). Immunol. 2005;174:1081-1090. ).

[0210] In some embodiments, the CoStARs of the invention comprise substantially all of the CD40 costimulatory domains. In some embodiments, the CoStARs of the invention comprise two or more CD40 costimulatory domains. In some embodiments, the CoStARs of the invention comprise a CD40 costimulatory domain signaling component, fragment, or motif. In some embodiments, the CD40 signaling fragment or motif comprises, consists of, or consists essentially of an SH3 binding sequence (e.g., but not limited to, KPTNKAPH (SEQ ID NO: 35), PTNKAPHP (SEQ ID NO: 443), or PTNKAPH (SEQ ID NO: 444)), a TRAF2 / TRAF3 binding sequence (e.g., but not limited to, 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., but not limited to, PQEINF (SEQ ID NO: 509), QEPQEINF (SEQ ID NO: 448), or QEPQEINFP (SEQ ID NO: 39)), or a PKA sequence (e.g., but not limited to, KKPTNKA (SEQ ID NO: 449)). NO: 40) or SRISVQE (SEQ ID NO: 41) and two or more, or three or more, or four or more such components or motifs or combinations thereof, which may be multiple copies and arranged in any order. In some embodiments, the CoStARs of the present invention comprise a CD40 costimulatory domain and a CD40 costimulatory domain signaling component or motif. In some embodiments, one or more of the SH3, RAF2 / 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 regulate proinflammatory and / or immunosuppressive cytokines. In some embodiments, adding tandem copies of those motifs and / or mutating certain motifs amplifies these effects.

[0211] Table 1 provides non-limiting examples of TRAF2 / TRAF3 binding domains. Table 1 Alignment with minor and major consensus sequences shown in Figures 82A-82B middle.

[0212] Thus, the TRAF2 / TRAF3 binding sequences of the present invention also include sequences such as P1V2Q3E4 and variants, wherein 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.

[0213] Illustrative, non-limiting examples of CD40 TRAF2 / TRAF3 sequence variants include amino acids at P-2, P-1, P0, and P1 enclosed by dashed lines below, and the TRAF2 / TRAF3-derived protein is identified.

[0214] Table 3 provides non-limiting examples of TRAF6 binding sequences. Table 3 has an alignment of the primary TRAF6 consensus sequences. Figure 82C shown.

[0215] Illustrative, non-limiting examples of CD40 TRAF6 sequence variants include amino acids at P-2, P-1, P0, P1, P2, and P3 enclosed by dashed lines below, and the TRAF6 sequence source is identified.

[0216] In some embodiments, the selection of one or more costimulatory domain signaling components or motifs is guided by the cell in which the CoStAR is expressed and / or the desired costimulatory activity more closely identified with the signaling component or motif, or avoiding costimulatory activity more closely identified with the signaling component or motif.

[0217] In some embodiments, the CoStAR signaling domain comprises, in addition to a CD40 costimulatory domain or signaling component or motif thereof, or two or more such domains or components or motifs, or a combination thereof, an additional full-length costimulatory domain or signaling component thereof from, 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.

[0218] For reference, the human CD28 protein sequence is shown by GenBank accession number NP_006130.1, including 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), which contains amino acids that constitute the antigen binding site and amino acids that form 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.

[0219] For reference, the human CD8 α chain protein sequence is shown by GenBank accession number NP_001139345.1, including a signal peptide (amino acid 1-21), an extracellular domain (amino acid 22-182), a transmembrane domain (amino acid 183-203) and a cytoplasmic domain (amino acid 204-235). The extracellular domain includes an immunoglobulin-type domain (amino acid 28-128), which contains amino acids constituting the antigen binding site and amino acids forming the homodimer interface. The extracellular domain includes several asparagine residues that can be glycosylated, and the intracellular domain includes serine and tyrosine residues that can be phosphorylated.

[0220] For reference, the human IgG4 constant region sequence is shown by UniProtKB / Swiss-Prot: Accession No. P01861.1, including CH1 (amino acids 1-98), hinge (amino acids 99-110), CH2 (amino acids 111-220), CH3 (amino acids 221-327). The CH2 region contains an asparagine at amino acid 177, which is glycosylated and is involved in Fc receptors and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0221] For reference, the protein sequence of human CD137 (4-1BB) (another TNFR superfamily member) is shown by GenBank accession number NP_001552.2, including a signal peptide (amino acid 1-23), an extracellular domain (amino acid 24-186), a transmembrane domain (amino acid 187-213) and a cytoplasmic domain (amino acid 214-255). The binding of CD137L ligand trimers expressed on antigen-presenting cells to CD137 leads to receptor trimerization and activation of signal 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).

[0222] For reference, the human CD134 (OX40) protein sequence is shown in GenBank accession number NP_003318.1, including 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). The receptor has been shown to activate NF-κB through its interaction with the adaptor proteins TRAF2 and TRAF5, and studies have shown that the receptor promotes the expression of the apoptosis inhibitors BCL2 and BCL21L1 / BCL2-XL.

[0223] Human T cell surface antigen CD2 has at least two isotypes. As a reference, the human CD2 isotype 1 protein sequence is shown by NP_001315538.1, including 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 isotype 2 protein sequence is shown by NP_001758.2.

[0224] For reference, the human CD357 (GITR) isoform-1 protein sequence is shown by GenBank Accession No. NP_004186.1, including 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).

[0225] For reference, the human CD29 (β1 integrin) protein sequence is shown by GenBank Accession No. NP_596867, including 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).

[0226] Human CD150 (SLAM) protein sequence has several isotypes. Except the transmembrane form of CD150 (mCD150), hematopoietic lineage cells express the mRNA of CD150 (sCD150) of coding secretory form, which lacks the whole transmembrane region of 30 amino acids. As a reference, human SLAM isotype b is shown by GenBank accession number NP_003028.1, including signal peptide (amino acid 1-20), extracellular domain (amino acid 21-237), transmembrane domain (amino acid 238-258) and cytoplasmic domain (amino acid 259-335). Human SLAM isotype a is shown by GenBank accession number NP_001317683.1.

[0227] CD278 or ICOS (inducible T cell co-stimulator) is a CD28 superfamily co-stimulatory molecule expressed on activated T cells. The human ICOS precursor (199 amino acids with a signal peptide) is shown by GenBank accession number NP_036224.1 and includes 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) is similar to certain binding sites of 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)).

[0228] Hematopoietic cell signal transducer (HCST), also known as DAP10, KAP10, PIK3AP, and hematopoietic cell signal transducer (HCST) (GenBank Accession No. 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 for the phosphatidylinositol 3-kinase binding site.

[0229] In an embodiment of the present invention, CoStAR can be expressed alone under the control of a promoter in a therapeutic cell population with therapeutic activity, such as tumor infiltrating lymphocytes (TIL). 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, such as 1, 2, 3, 4, 5 or up to 10 amino acids may be missing at the N-terminus of the mature receptor protein). Therefore, in one aspect, the present invention also relates to CoStAR constructs, which are not limited to those having a sequence shown in any one of SEQ ID NOs: 42-185, 192-335, 344-430, including one of these sequences lacking up to about 5, such as 1, 2, 3, 4, 5 or up to 10 amino acids at the N-terminus of the mature receptor protein. Suitable TCRs and CARs are well known in the literature, 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), which have been successfully used to treat myeloma or B cell malignancies, respectively. The CoStARs described herein can be expressed with any known CAR or TCR, thereby providing cells with an adjustable growth switch to allow cells to expand in vitro or in vivo, as well as conventional activation mechanisms in the form of TCRs or CARs for anticancer activity. Therefore, the present invention provides cells for adoptive cell therapy, comprising CoStARs as described herein and TCRs and / or CARs that specifically bind to tumor-associated antigens. Exemplary CoStARs comprising CD28 include an extracellular antigen binding domain and extracellular, transmembrane, and intracellular signaling domains.

[0230] The term "antigen binding domain" as used herein refers to an antibody fragment, including but not limited to diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen binding domain is capable of binding to the same antigen bound by a parent antibody or parent antibody fragment (e.g., a parent scFv). In some embodiments, an antigen binding fragment may comprise one or more complementary determining regions (CDRs) from a specific human antibody grafted onto a framework (FR) from one or more different human antibodies. An "antigen binding domain" may be referred to as a "ligand binding domain".

[0231] Antigen binding domains can be prepared to be 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. TAA 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. TAA also includes neoantigens, peptide / MHC complexes and HSP / peptide complexes.

[0232] In some embodiments, the antigen binding domain comprises a T cell receptor or a binding fragment thereof that binds to a determined tumor-specific peptide-MHC complex. The term "T cell receptor" or "TCR" refers to a heterodimeric receptor composed of αβ or γδ chains that are paired on the surface of T cells. Each α, β, γ, and δ chain consists of two Ig-like domains: a variable domain (V) that confers antigen recognition through a complementary determining region (CDR), followed by a constant domain (C) that is anchored to the cell membrane through 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 between 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, 868p.).

[0233] In some embodiments, the antigen binding domain comprises a natural ligand of a tumor-expressed protein or its tumor-binding fragment. A non-limiting example is PD1 combined with PDL1. Another example is transferrin receptor 1 (TfR1), also known as CD71, a homodimeric protein of a key regulator of cellular iron homeostasis and proliferation. Although TfR1 is expressed at low levels in a variety of cells, it is expressed at higher levels in rapidly proliferating cells (including malignant cells where overexpression is associated with poor prognosis). In some embodiments of the present invention, the antigen binding domain comprises transferrin or its transferrin receptor binding fragment.

[0234] 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 may 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.

[0235] In various embodiments, the present invention provides a CoStAR comprising:

[0236] i. a scFv that binds to carcinoembryonic antigen (CEA), a spacer region, and the transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain;

[0237] ii. scFv that binds to CEA, the spacer and transmembrane sequence of CD28, and the CD40 signaling domain;

[0238] iii. scFv that binds to CEA, a spacer and the transmembrane sequence of CD28, a CD137 signaling domain, and a CD40 signaling domain;

[0239] iv. scFv that binds to CEA, a spacer, and the transmembrane sequence of CD28, a CD134 signaling domain, and a CD40 signaling domain;

[0240] v. scFv that binds to CEA, a spacer, and the transmembrane sequence of CD28, a CD2 signaling domain, and a CD40 signaling domain;

[0241] vi. scFv that binds to CEA, a spacer and the transmembrane sequence of CD28, a GITR signaling domain, and a CD40 signaling domain;

[0242] vii. scFv that binds to CEA, a spacer and the transmembrane sequence of CD28, a CD29 signaling domain, and a CD40 signaling domain;

[0243] viii. scFv that binds to CEA, a spacer and the transmembrane sequence of CD28, a CD150 signaling domain, and a CD40 signaling domain;

[0244] ix. scFv that binds to CEA, a spacer, and the transmembrane sequence of CD8, a CD28 signaling domain, and a CD40 signaling domain;

[0245] x. scFv that binds to CEA, the spacer and transmembrane sequence of CD8, and the CD40 signaling domain;

[0246] xi. scFv that binds to CEA, a spacer and the transmembrane sequence of CD8, a CD137 signaling domain, and a CD40 signaling domain;

[0247] xii. scFv that binds to CEA, a spacer and the transmembrane sequence of CD8, a CD134 signaling domain, and a CD40 signaling domain;

[0248] xiii. scFv that binds to CEA, a spacer and the transmembrane sequence of CD8, a CD2 signaling domain, and a CD40 signaling domain;

[0249] xiv. scFv that binds to CEA, a spacer and the transmembrane sequence of CD8, a GITR signaling domain, and a CD40 signaling domain;

[0250] xv. scFv that binds to CEA, a spacer and the transmembrane sequence of CD8, a CD29 signaling domain, and a CD40 signaling domain;

[0251] xvi. scFv that binds to CEA, a spacer and the transmembrane sequence of CD8, a CD150 signaling domain, and a CD40 signaling domain;

[0252] xvii. a 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;

[0253] xviii. a scFv that binds to CEA, a spacer region comprising an IgG4 constant region, a CD28 transmembrane sequence, and a CD40 signaling domain;

[0254] xix. a scFv that binds to CEA, a spacer region comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD137 signaling domain, and a CD40 signaling domain;

[0255] xx. a scFv that binds to CEA, a spacer region comprising an IgG4 constant region and a CD28 transmembrane sequence, a CD134 signaling domain, and a CD40 signaling domain;

[0256] xxi. a 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;

[0257] xxii. a scFv that binds to CEA, a spacer region comprising an IgG4 constant region and a CD28 transmembrane sequence, a GITR signaling domain, and a CD40 signaling domain;

[0258] xxiii. a scFv that binds to CEA and comprises a spacer region of an IgG4 constant region and a CD28 transmembrane sequence, a CD29 signaling domain, and a CD40 signaling domain;

[0259] xxiv. a 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;

[0260] xxv. a 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 CD40 signaling domain;

[0261] xxvi. a 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 mutated CD40 signaling domain;

[0262] xxvi. Binds to the PDL1 binding domain, short spacer, and transmembrane sequence of CD28, CD28 signaling domain, and CD40 signaling domain;

[0263] xxvi. Binds to the PDL1 binding domain, short spacer, and transmembrane sequence of CD28 and the CD40 signaling domain;

[0264] xxix. a binding domain that binds to CD155, CD112, or CD113, a CD28 transmembrane domain, a CD28 signaling domain, and a CD40 signaling domain;

[0265] xxx. A binding domain for CD155, CD112, or CD113, a CD28 transmembrane domain, and a CD40 signaling domain;

[0266] xxxi. scFv that binds to CEA, a binding domain that binds to PDL1, a short spacer and a transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain;

[0267] xxxii. 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;

[0268] xxxiii. a scFv that binds to CEA, a binding domain that binds to CD155, CD112, or CD113, a short spacer and a transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain;

[0269] xxxiv. scFv that binds to CEA, a binding domain for CD155, CD112, or CD113, a short spacer and transmembrane sequence of CD28, and a CD40 signaling domain;

[0270] xxxv. scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28, a transmembrane sequence of CD28, and an NTRK1 signaling domain;

[0271] xxxvi. a scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, an NTRK1 signaling domain, and a CD40 signaling domain;

[0272] xxxvii. A scFv that binds to CEA, comprises a spacer region of the extracellular domain of CD28 and a transmembrane sequence of CD28, a CD28 signaling domain, and an NTRK1 signaling domain;

[0273] xxxviii. a scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, an NTRK1 signaling domain, and a CD40 signaling domain;

[0274] xxxix. A scFv that binds to CEA, a spacer region comprising the ICOS extracellular domain and the ICOS transmembrane sequence, an ICOS signaling domain, and a CD40 signaling domain;

[0275] xl. scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, and an ICOS signaling domain;

[0276] xli. scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, an ICOS signaling domain, and a CD40 signaling domain;

[0277] xlii. scFv that binds to CEA, comprising a spacer region of the CD2 extracellular domain and a transmembrane sequence of CD2, a CD2 signaling domain, and a CD40 signaling domain;

[0278] xliii. a scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, and a CD2 signaling domain;

[0279] xliv. A scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, a CD40 signaling domain, and a CD2 signaling domain;

[0280] xlv. a scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, and a CD137 signaling domain;

[0281] xlvi. a scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, a CD40 signaling domain, and a CD137 signaling domain;

[0282] xlvi. scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, and a DAP10 signaling domain;

[0283] xlviii. a scFv that binds to CEA, a spacer comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, a CD40 signaling domain, and a DAP10 signaling domain;

[0284] xlix. scFv that binds to CEA, a spacer region comprising the extracellular domain of CD28 and the transmembrane sequence of CD28, a CD28 signaling domain, and a CD134 signaling domain;

[0285] xlx. scFv that binds to CEA, comprises a spacer region of the CD28 extracellular domain and the transmembrane sequence of CD28, a CD40 signaling domain, and a CD134 signaling domain.

[0286] In some embodiments, the present invention provides a CoStAR comprising a scFv that binds to MSLN, wherein the scFv is structurally linked to the spacer, transmembrane, and signaling domains of any one of paragraphs i-xlx.

[0287] In some embodiments, the present invention provides a CoStAR comprising a scFv that binds to FolR1, wherein the scFv is structurally linked to the spacer, transmembrane, and signaling domains of any one of paragraphs i-xlx.

[0288] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxxiv, and binding to FolR1 through a binding domain comprising an antigen-binding fragment of scFv MOV19 (SEQ ID NO: 9).

[0289] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxxiv and binds to CEA through a binding domain comprising an antigen-binding fragment of scFvMFE23 (SEQ ID NO: 10).

[0290] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binds to CEA through a binding domain of an antigen-binding fragment comprising scFv MFE23 (K>Q) (SEQ ID NO: 11).

[0291] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binds to CEA through a binding domain comprising an antigen-binding fragment of humanized scFv MFE23 (hMFE23) (SEQ ID NO: 12).

[0292] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binding to CEA through a binding domain comprising an antigen-binding fragment of scFv CEA6 (SEQ ID NO: 13).

[0293] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binding to CEA through a binding domain comprising an antigen-binding fragment of scFv BW431 / 26 (SEQ ID NO: 14).

[0294] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binding to CEA through a binding domain comprising an antigen-binding fragment of scFv HuT84.66 (M5A) (SEQ ID NO: 15).

[0295] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxxiv, and binding to FolR1 through a binding domain comprising an antigen-binding fragment of scFv MOV19 (SEQ ID NO: 9).

[0296] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxxiv and xxxi-xxxiv, and binds to MSLN through a binding domain of an antigen-binding fragment comprising scFv SS1 (SEQ ID NO: 186).

[0297] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binds to MSLN through a binding domain of an antigen-binding fragment comprising scFv M5 (humanized SS1) (SEQ ID NO: 187).

[0298] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binding to MSLN through a binding domain comprising an antigen-binding fragment of humanized scFv HN1 (SEQ ID NO: 188).

[0299] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binds to MSLN through a binding domain of an antigen-binding fragment comprising scFv M912 (SEQ ID NO: 189).

[0300] In some embodiments, the present invention provides a CoStAR comprising the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binding to MSLN through a binding domain comprising an antigen-binding fragment of scFv HuYP218 (SEQ ID NO: 190).

[0301] In some embodiments, the present invention provides CoStAR, which comprises the spacer, transmembrane and signaling domain structures of any one of i-xxvi and xxxi-xxxiv, and binds to MSLN through a binding domain of an antigen-binding fragment comprising scFv P4 (SEQ ID NO: 191).

[0302] In some embodiments, any of the above (including I-xlx) can be used with CD40, TRAF2, TRAF2 / 3 or TRAF6 variants provided herein, including Figure 28 、 67 , 77A-77C, 83-89D and 92 (excluding Figure 78 and 79 ), and including those in Tables 2, 4-5, and 7-14, and including SEQ ID NOs: 42, 475-479, 494-498, 505, 510-519, and 840-40839, and consensus sequences AC.

[0303] As used herein, the terms "specifically bind" or "specific for" refer to a measurable and reproducible interaction, such as 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 biomolecules. For example, an antibody portion that specifically binds to a target (which can 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 a binding affinity that is at least about 10 times greater than its binding affinity for other targets to one or more antigenic determinants of the antigen (e.g., a cell surface antigen or a peptide / MHC protein complex). spacer

[0304] The CoStAR of the present invention is optionally included in 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 close to the target antigen and receptor ligand. In some embodiments, a long spacer is used, for example, to target membrane proximal epitopes or glycosylated antigens (see Guest RD et al., The role of extracellular spacer regions in the optimal design of chimeric immunereceptors: evaluation of four different scFvs and antigens. J. Immunother. 2005; 28: 203-211; Wilkie S. et al., Retargeting of human T cells to tumor-associatedMUC1: the evolution of a chimeric antigen receptor. J. Immunol. 2008; 180: 4901-4909). In other embodiments, CoStARs have short spacers, for example, to target membrane-distal epitopes (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 spacerdomain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol. Res. 2015; 3: 125-135). In some embodiments, the spacer comprises all or part of an IgG hinge 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 a spacer that comprises an insertion, deletion, or mutation in an IgG hinge. In some embodiments, the spacer may comprise all or part of one or more antibody constant domains, such as, but not limited to, a CH2 and / or CH3 domain.In some embodiments, in a spacer comprising all or part of a CH2 domain, the CH2 domain is modified so as not to bind to an Fc receptor. For example, it has been found that Fc receptor binding in myeloid cells impairs CAR T cell function. In some embodiments, the spacer comprises all or part of an Ig-like hinge from CD28, CD8, or other proteins comprising a hinge region. In some embodiments of the invention comprising a spacer, the length of the spacer is 1 to 50 amino acids.

[0305] In one non-limiting embodiment, the spacer comprises substantially all of the extracellular domain, such as the CD28 extracellular domain (i.e., about amino acids 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, such as a portion of the CD28 extracellular domain, and may lack all or most of the Ig domain. In another embodiment, the spacer comprises amino acids from about 141 to about 152 of CD28, but does not comprise the remainder of the CD28 extracellular domain. In another embodiment, the spacer comprises amino acids from about 128 to about 182 of CD8, but does not include the remainder of the CD8 extracellular domain. connector

[0306] In some embodiments, the CoStAR extracellular domain comprises a linker. The linker comprises a short chain of amino acids for connecting a domain, such as a binding domain or a transmembrane domain with a spacer. In order to have flexibility in binding to a ligand, the ligand binding domain is typically connected to the spacer or transmembrane domain via a flexible linker comprising about 5 to 25 amino acids, such as, for example, AAAGSGGSG (SEQ ID NO: 18), GGGGSGGGGSGGGGS (SEQ ID NO: 431). In some embodiments, the CoStAR comprises a binding domain directly connected to the transmembrane domain via a linker and has no spacer. In some embodiments, the CoStAR comprises a binding domain directly connected to the transmembrane domain via a spacer and has no linker. signaling domain

[0307] As described above, in some embodiments, CoStAR comprises a full-length primary co-stimulatory receptor, which may include, but is not limited to, an extracellular ligand binding and 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 co-stimulatory receptor comprises a chimeric protein, for example, comprising an extracellular ligand binding domain of one of the above proteins and an intracellular signaling domain of another of the above 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), 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 co-stimulatory receptors are capable of dimerization. Without being bound by theory, it is believed that CoStAR dimerizes or associates with other accessory molecules to initiate a signal. In some embodiments, CoStAR dimerizes or associates with accessory molecules through transmembrane domain interactions. In some embodiments, dimerization or association with accessory molecules is assisted by co-stimulatory receptor interactions in the intracellular portion and / or extracellular portion of the co-stimulatory receptor. transmembrane domain

[0308] Although the main function of the transmembrane is to anchor CoStAR in the T cell membrane, in some embodiments, the transmembrane domain affects CoStAR function. In some embodiments, the transmembrane domain is composed of a full-length primary costimulatory receptor domain. In the present invention, the CoStAR construct includes an extracellular domain of a receptor and an intracellular signaling domain of a second receptor, and the transmembrane domain can be a transmembrane domain of an extracellular domain or an intracellular domain. In some embodiments, the transmembrane domain is from CD4, CD8α, CD28 or ICOS. Gueden et al. associated the use of the ICOS transmembrane domain with increased CAR T cell persistence and overall anti-tumor efficacy (Guedan S. et al., Enhancing CART cell persistence through ICOS and 4-1BB costimulation. JCI Insight. 2018; 3: 96976). In some embodiments, the transmembrane domain includes a hydrophobic alpha helix across the cell membrane.

[0309] 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 CoStARs of the present invention may include several amino acids between the transmembrane domain and the signaling domain. For example, in one construct described herein, the connection 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). Variants

[0310] In some embodiments, the amino acid sequence variants of the antibody portion or other portions provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody portion. The amino acid sequence variants of the antibody portion can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody portion or by peptide synthesis. Such modifications include, for example, the disappearance of the amino acid sequence of the antibody portion, and / or are inserted into the amino acid sequence of the antibody portion, and / or replace the amino acid sequence of the antibody portion. Any combination of disappearance, insertion, and substitution can be performed to obtain the final construct, provided that the final construct has desired characteristics, such as antigen binding.

[0311] In some embodiments, an antibody binding domain portion comprising one or more amino acid substitutions, deletions or insertions is provided. The target sites of the mutation change include the antibody binding domain heavy chain and light chain variable regions (VR) and framework (FR). Amino acid substitutions can be introduced into the target binding domain and the product of the desired activity can be screened, such as retained / improved antigen binding or reduced immunogenicity. In some embodiments, amino acid substitutions can be introduced into one or more primary co-stimulatory receptor domains (extracellular or intracellular), secondary co-stimulatory receptor domains or extracellular co-receptor domains. Therefore, the present invention covers the CoStAR proteins and components specifically disclosed herein and their variants, i.e., CoStAR proteins and components 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%, at least 99% sequence identity to the amino acid sequences specifically disclosed herein. When referring to specific sequences, the terms "percent similarity," "percent identity," and "percent homology" are used as indicated in the University of Wisconsin GCG software program BestFit. Other algorithms, such as BLAST, psiBLAST, or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), can be used.

[0312] Specific amino acid sequence variants may differ from a reference sequence by insertion, addition, substitution, or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20, or 20-30 amino acids. In some embodiments, a variant sequence may comprise a reference sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more inserted, deleted, or substituted residues. For example, 5, 10, 15, up to 20, up to 30, or up to 40 residues may be inserted, deleted, or substituted.

[0313] In some preferred embodiments, variants can differ from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. Conservative substitutions include replacing amino acids with different amino acids having similar properties. For example, an aliphatic residue can be replaced by another aliphatic residue, a non-polar residue can be replaced by another non-polar residue, an acidic residue can be replaced by another acidic residue, a basic residue can be replaced by another basic residue, a polar residue can be replaced by another polar residue, or an aromatic residue can be replaced by another aromatic residue. Conservative substitutions can, for example, be made between the amino acids in the following groups.

[0314] Conservative substitutions are shown in Table 6 below.

[0315] Amino acids can be divided into different classes based on 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 affecting chain orientation: Gly, Pro; Aromatic: Trp, Tyr, Phe. Non-conservative substitutions would involve replacing a member of one of these classes with another. cell

[0316] The cells used in the present invention may 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 may be allogeneic or autologous to the patient.

[0317] T cells or T lymphocytes are lymphocytes that play an important 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 associated with transplant rejection. CTLs express CD8 molecules on their surface.

[0318] These cells recognize their targets by binding to antigens present on the surface of all nucleated cells in association with MHC class I. IL-10, adenosine, and other molecules secreted by regulatory T cells can inactivate CD8+ cells into an anergic state, preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0319] Memory T cells are a subpopulation of antigen-specific T cells that persist for a long time after the infection resolves. After being exposed to their cognate antigens again, they rapidly expand into a large number of effector T cells, thereby providing the immune system with "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two effector memory T cells (TEM cells and TEMRA cells). Memory cells can be 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 essential for maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity at the end of the immune response and to inhibit self-reactive T cells that escape the negative selection process in the thymus.

[0320] Two main categories of CD4+ Treg cells have been described—naturally occurring Treg cells and adaptive Treg cells. Naturally occurring Treg cells (also known as CD4 + CD25 + FoxP3 + Treg cells (Treg cells) appear in the thymus and have been associated with the interaction between developing T cells and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. 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 cells or Th3 cells) can be generated during normal immune responses.

[0321] 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 innate lymphoid cell group) are defined as large granular lymphocytes (LGLs) and constitute the third type of cell that differentiates from the common lymphoid progenitor cell that gives rise to B and T lymphocytes.

[0322] 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. Due to TCR-mediated recognition of receptor alloantigens, autologous cells expressing CoStAR may be advantageous in avoiding graft-versus-host disease (GVHD). In addition, the immune system of the CoStAR receptor can attack the infused CoStAR cells, causing rejection. In certain embodiments, in order to prevent GVHD and reduce rejection, endogenous TcR is removed from allogeneic CoStAR cells by genome editing. Nucleic Acids

[0323] One aspect of the present invention provides a nucleic acid sequence of the present invention that encodes any CoStAR, polypeptide, or protein described herein (including functional portions and functional variants thereof). As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it should be understood that those skilled in the art can use conventional techniques to make nucleotide substitutions without affecting the polypeptide sequence encoded by the polynucleotide to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed, such as codon optimization. Nucleic acids according to the present invention can comprise DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides in which synthetic or modified nucleotides are included. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, 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 should be understood that polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0324] The terms "variant," "homologue," or "derivative" in relation to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleic acid(s) to or from the sequence.

[0325] The nucleic acid sequence may encode a CoStAR protein, including but not limited to any one of SEQ ID NOs: 42-247 or variants thereof. The nucleotide sequence may comprise a codon-optimized nucleic acid sequence as shown, engineered for expression in human cells.

[0326] The present invention also provides a nucleic acid sequence comprising a nucleic acid sequence encoding CoStAR and other nucleic acid sequences encoding a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0327] Nucleotide sequence can be connected by the sequence that allows two or more nucleic acid sequences to be co-expressed.For example, construct can comprise internal promoter, internal ribosome entry sequence (IRES) or the sequence encoding cleavage site.The cleavage site can be self-cleavage, so that when producing polypeptide, it is cut into separated protein immediately, and does not need any external cleavage activity.Known various self-cleavage sites, comprise foot-and-mouth disease virus (FMDV) and 2A self-cleavage peptide.The co-expression sequence can be internal ribosome entry sequence (IRES).The co-expression sequence can be internal promoter. carrier

[0328] In one aspect, the present invention provides a vector comprising the nucleic acid sequence or nucleic acid construct of the present invention.

[0329] This vector can be used to introduce a nucleic acid sequence or nucleic acid construct into a host cell to express one or more CoStARs according to the first aspect of the present invention and optionally one or more other proteins of interest (POIs), such as TCRs or CARs. The vector can be, for example, a plasmid or viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or a synthetic mRNA.

[0330] The nucleic acids of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patents Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0331] Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of one or more transgenics and their proliferation in daughter cells. The vector may be able to transfect or transduce lymphocytes including T cells or NK cells. The present invention also provides vectors into which the nucleic acid of the present invention has been inserted. The expression of natural or synthetic nucleic acids encoding CoStAR and optionally TCR or CAR is typically achieved by operably connecting the nucleic acid encoding CoStAR and TCR / CAR polypeptides or portions thereof to one or more promoters and incorporating the construct into an expression vector.

[0332] Other 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 recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible so that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline.

[0333] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongated growth factor-1α (EF-1α). However, other constitutive promoter sequences may 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 (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.

[0334] The vector may be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of desired nucleic acid sequences. Viral vector technology is well known in the art and is described 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 WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193). In some embodiments, the construct expressed is as shown in SEQ ID NO: 42-247. In some embodiments, the nucleic acid is a polycistronic construct that allows the expression of multiple transgenes (e.g., CoStAR and TCR and / or CAR, etc.) under the control of a single promoter. In some embodiments, transgenes (e.g., CoStAR and TCR and / or CAR, etc.) are separated by self-cleavage 2A peptides. Examples of 2A peptides that can be used for nucleic acid constructs of the present invention include F2A, P2A, T2A, and E2A. In other embodiments of the present invention, the nucleic acid construct of the present invention is a polycistronic construct comprising two promoters; one promoter drives the expression of CoStAR, while the other promoter drives the expression of TCR or CAR. 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. In order to evaluate the expression of the CoStAR polypeptide or portion thereof, the expression vector introduced into the cell may also contain a selective marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population sought to be transfected or transduced by a viral vector. Cell source

[0335] Before amplification and genetic modification, a cell source (e.g., immune effector cells, such as T cells or NK cells) is obtained from a subject. The term "subject" is intended to include a living organism (e.g., mammal) that can induce an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. 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 infection site, ascites, pleural effusion, spleen tissue, and tumors.

[0336] In one aspect, T cells are separated from peripheral blood lymphocytes by lysing red blood cells and depleting mononuclear cells, for example, by PERCOLL™ gradient centrifugation or by countercurrent centrifugal elution. T cells can be collected at apheresis centers and cell storage facilities where they can be harvested, maintained, and easily transferred. T cells can be cryopreserved and stored for future use. An acceptable storage duration can be determined and verified, and the acceptable storage duration can be as long as 6 months, as long as one year, or longer.

[0337] In some embodiments, tumor infiltrating cells (TIL) are separated and / or amplified from a tumor, for example, by fragmentation, incision, or enzymatic digestion of a tumor biopsy or a mass. Tumor biopsy can be used as starting material to produce TIL in a two-stage process: stage 1 (typically performed for 2-3 hours) uses incision, enzymatic digestion, and homogenization to initially collect and process the tumor material to produce a single cell suspension, which can be directly cryopreserved to stabilize the starting material for subsequent manufacture, and stage 2, which can occur after a few days or years. Stage 2 can be carried out for 4 weeks, which can be a continuous process, starting from thawing the product of stage 1 and culturing TIL (about 2 weeks) from the tumor starting material, followed by a rapid expansion process of TIL cells (about 2 weeks), to increase the amount of cells and therefore increase the dosage. Concentrated and washed TIL can be prepared before the liquid suspension of cells.

[0338] TIL colonies can be transduced at any point after collection. In some embodiments, cryopreserved TIL colonies are transduced to express CoStAR after thawing. In some embodiments, TIL colonies are transduced to express CoStAR during the growth or initial expansion of tumor starting material. In some embodiments, TIL colonies are transduced to express CoStAR during REP (such as, but not limited to, REP from about 8 days to about 10 days). Exemplary TIL preparations are described in U.S. patent application serial number 62 / 951,559 of the applicant, filed December 20, 2019.

[0339] Specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, by using beads conjugated with anti-CD3 / anti-CD28 (e.g. In one embodiment, the incubation time period of the M-450 CD3 / CD28 beads is sufficient to separate the T cells required for positive selection. In one aspect, the incubation time period is about 30 minutes. In other aspects, the scope of the incubation time period is 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In other aspects, the incubation time period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred aspect, the incubation time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Compared with other cell types, in any case where T cells are rare, for example, when separating tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals, longer incubation times can be used to separate T cells. In addition, longer incubation times can be used to improve the capture efficiency of CD8+T cells. Therefore, by simply shortening or extending the time allowing T cells to bind to the CD3 / CD28 beads and / or by increasing or reducing the ratio of beads to T cells (as further described herein), T cell subsets can be preferentially selected at the beginning of cultivation or at other time points in the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be preferentially selected at the beginning of cultivation or at other required time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some aspects, it may be necessary to perform a selection process and use "unselected" cells in activation and amplification processes. Further selection rounds can also be performed on "unselected" cells.

[0340] Enrichment of T cell colonies by negative selection can be achieved with a combination of antibodies for surface markers unique to negative selection cells. One method is to carry out cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, which uses a monoclonal antibody mixture for the cell surface markers present on the cells of negative selection. For example, in order to enrich CD4+ cells by negative selection, monoclonal antibody mixtures generally include antibodies for CD14, CD20, CD16, HLA-DR and CD8. In some aspects, it may be necessary to enrich or positively select regulatory T cells that generally express CD4+, CD25+, CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150 and FoxP3+. Alternatively, in some aspects, regulatory T cells are exhausted by beads or other similar selection methods conjugated with anti-CD25.

[0341] Methods described herein may include, for example, using, for example, negative selection techniques as described herein to select a specific subpopulation of immune effector cells (such as T cells), wherein the immune effector cells are colonies exhausted by regulatory T cells, the cells exhausted by CD25+. Preferably, the cell colony exhausted by regulatory T cells contains and is less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0342] Specific subpopulations of CoStAR effector cells that specifically bind to the target antigen can be enriched by positive selection techniques. For example, in some embodiments, the effector cells are enriched by incubating the abTCR effector cells required for positive selection with beads conjugated to the target antigen for a period of time. In some embodiments, the period is about 30 minutes. In some embodiments, the range of the period is 30 minutes to 36 hours or longer (including all ranges between these values). In some embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. In order to separate the effector cells present at low levels in the heterogeneous cell population, a longer incubation time, such as 24 hours, can increase cell yield. Compared to other cell types, in any case where there are few effector cells, a longer incubation time can be used to separate the effector cells. Skilled technicians will recognize that multiple rounds of selection can also be used in the context of the present invention.

[0343] The T cells used for stimulation can also be frozen after the washing step. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and are useful in this context, one method includes using PBS containing 20% ​​DMSO and 8% human serum albumin, or 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 culture medium or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1° / minute and storing them in the vapor phase of a liquid nitrogen storage tank. Controlled freezing and other methods of uncontrolled freezing at -20°C or in liquid nitrogen can be used. Allogeneic CoStAR

[0344] In embodiments described herein, immune effector cells can be allogeneic immune effector cells, such as T cells or NK cells.For example, cells can be allogeneic T cells, such as allogeneic T cells lacking endogenous T cell receptors (TCR) and / or human leukocyte antigens (HLA) (such as HLA class I and / or HLA class II) expression.

[0345] The T cell lacking functional endogenous TCR can be, for example, engineered so that it does not express any functional TCR on its surface, engineered so that it does not express one or more subunits comprising functional TCR (e.g., engineered so that it does not express (or exhibits reduced expression) TCR α, TCR β, TCR γ, TCR δ, TCR ε and / or TCR ζ) or engineered so that it produces very little functional TCR on its surface. Alternatively, T cells can express significantly impaired TCR, for example, by expressing a mutation or truncated form of one or more subunits of TCR. The term "significantly impaired TCR" means that the TCR does not elicit an adverse immune response in the host.

[0346] The T cells described herein can, for example, be engineered so that they do not express functional HLA on their surface. For example, the T cells described herein can be engineered so that the cell surface expresses HLA, such as HLA class I and / or HLA class II, which are downregulated. In some aspects, downregulation of HLA can be achieved by reducing or eliminating the expression of beta-2 microglobulin (B2M).

[0347] In some embodiments, T cells may lack functional TCR and functional HLA, such as HA class I and / or HLA class II. Modified T cells lacking functional TCR and / or HLA expression can be obtained by any suitable means, including knocking out or knocking down one or more subunits of TCR or HLA. For example, T cells can include knocking down 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).

[0348] In some embodiments, allogeneic cells can be cells that do not express or express inhibitory molecules at low levels, such as cells engineered by any method described herein. For example, cells can be cells that do not express or express inhibitory molecules at low levels, such as cells that can reduce the ability of cells expressing CoStAR to cause immune effector responses. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (such as 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 TGFRβ. The inhibition of inhibitory molecules, such as by inhibition at the DNA, RNA or protein level, can optimize the performance of cells expressing CAR. In embodiments, inhibitory nucleic acids, such as inhibitory nucleic acids, such as dsRNA, such as siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN) or zinc finger endonuclease (ZFN), such as described herein, can be used.

[0349] Use of siRNA or shRNA to inhibit endogenous TCR or HLA

[0350] In some embodiments, TCR expression and / or HLA expression in T cells can be inhibited using siRNA or shRNA targeting nucleic acids encoding TCR and / or HLA and / or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (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 TGFRβ).

[0351] Expression of siRNA and shRNA in T cells can be achieved using any conventional expression system, such as, for example, a lentiviral expression system. Exemplary shRNAs that downregulate expression of TCR components are described, for example, in U.S. Publication No. 2012 / 0321667. Exemplary siRNAs and shRNAs that downregulate expression of HLA class I and / or HLA class II genes are described, for example, in U.S. Publication No. US 2007 / 0036773.

[0352] CRISPR inhibition of TCR or HLA

[0353] As used herein, "CRISPR" or "CRISPR that inhibits TCR and / or HLA" refers to a set of clustered regularly interspaced short palindromic repeats or a system comprising such a set of repeats. As used herein, "Cas" refers to a CRISPR-associated protein. A "CRISPR / Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate the TCR and / or HLA genes described herein, and / or the present inhibitory molecules (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (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 TGFRβ).

[0354] Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8:172. This system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315:1709-1712; Marragini et al. (2008) Science 322:1843-1845.

[0355] T cell activation and expansion

[0356] T cells can generally be activated and expanded using methods such as those described in U.S. Patents 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.

[0357] Typically, the T cells of the present invention can be amplified by being attached with a reagent stimulating CD3 / TCR complex related signals and a surface contact of a part stimulating a co-stimulatory molecule on the T cell surface. In particular, it can be as described herein, such as by contacting with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody fixed on the surface, or by contacting with a protein kinase C activator (such as bryostatin) in conjunction with a calcium ion carrier, stimulating T cell colonies. In order to costimulate auxiliary molecules on the T cell surface, a part in conjunction with auxiliary molecules is used. For example, T cell colonies can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can be used as generally known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0358] In some embodiments, the expansion can be carried out using a flask or container or a breathable container known to those skilled in the art, and can be carried out 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 be carried out for about 14 days.

[0359] In some embodiments, expansion can be performed using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulators can include, for example, anti-CD3 antibodies, such as about 30 ng / ml OKT3, mouse monoclonal anti-CD3 antibodies (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 (including antigenic portions thereof, such as epitopes), which can be optionally expressed by a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, for example, 0.3 uM MART-1: 26-35 (27L) or gp100: 209-217 (210M), in the presence of a T cell growth factor (e.g., 300 IU / mL IL-2 or IL-15). Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. CoStAR cells can also be rapidly expanded by re-stimulation with the same cancer antigen pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, CoStAR cells can be further stimulated with, for example, irradiated autologous lymphocytes, or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, as exemplified. 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.

[0360] 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 70 In some embodiments, the amount of IL-2 in the dosage form may be about 100 IU / mL, about 7500 IU / mL, or about 8000 IU / mL, or 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL.

[0361] In some embodiments, the cell culture medium comprises the 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.

[0014] In some embodiments, the OKT3 antibody is administered at a concentration of about 1 μg / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, about 1 μg / mL, or 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL.

[0362] In some embodiments, the combination of IL-2, IL-7, IL-15 and / or IL-21 is used as a combination during amplification. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 and any combination thereof may be included during amplification. In some embodiments, the combination of IL-2, IL-15 and IL-21 is used as a combination during amplification. In some embodiments, IL-2, IL-15 and IL-21 and any combination thereof may be included.

[0363] In some embodiments, expansion can be performed in a supplemented cell culture medium comprising IL-2, OKT-3, and antigen presenting feeder cells.

[0364] In some embodiments, the expansion medium comprises 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 of IL-15, or about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15, or about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15, or about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15, or about 200 IU / mL of IL-15, or about 180 IU / mL of IL-15.

[0365] In some embodiments, the expansion medium comprises 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 20 1 IU / mL of IL-21 to about 0.5 IU / mL of IL-21, or about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21, or about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21, or about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21, or about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21, or about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21.

[0366] 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 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500, or 1 to 50 and 1 to 300, or 1 to 100 and 1 to 200.

[0367] In some aspects, the primary stimulation signal and the costimulatory signal of T cell can be provided by different schemes.For example, the reagent providing each signal can be in solution or coupled to a surface.When coupled to a surface, reagent can be coupled to identical surface (i.e., with " cis " form) or separated surface (i.e., with " trans " form). Alternatively, a kind of reagent can be coupled to a surface, and another kind of reagent can be in solution. In one aspect, the reagent providing costimulatory signal is combined with the cell surface, and the reagent providing primary activation signal is in solution or coupled to a surface. In some aspects, two kinds of reagents can be in solution. In one aspect, reagent can be a soluble form, which is then cross-linked to a surface, such as expressing a cell or antibody of an Fc receptor or by other binding agents of a binding agent. In this regard, referring to, for example, the artificial antigen presenting cells (aAPC) of U.S. Patent Application Publication Nos. 20040101519 and 20060034810, it is contemplated that the T cell in the present invention is activated and amplified.

[0368] In one aspect, two reagents are immobilized on the beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). For example, the reagent that provides the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the reagent that provides the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and the two reagents are co-immobilized on the same bead at equal molecular weight. In one aspect, for CD4+ T cell expansion and T cell growth, a 1:1 ratio of each antibody is used to bind to the beads. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies is used to bind to the beads such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In a specific aspect, an increase of about 1 to about 3 times is observed compared to the expansion observed using a 1:1 ratio. In one aspect, the ratio of CD3:CD28 antibodies to the beads ranges from 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the CD3:CD28 ratio is less than 1. In certain aspects of the invention, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to the beads is greater than 2:1. In a specific aspect, a 1:100 CD3:CD28 ratio of antibodies is used for binding to the beads. In one aspect, a 1:75 CD3:CD28 ratio of antibodies is used for binding to the beads. In other aspects, a 1:50 CD3:CD28 ratio of antibodies is used for binding to the beads. In one aspect, a 1:30 CD3:CD28 ratio of antibodies is used for binding to the beads. In a preferred aspect, a 1:10 CD3:CD28 ratio of antibodies is used for binding to the beads. In one aspect, a 1:3 CD3:CD28 ratio of antibodies is used for binding to the beads. In one aspect, a 3:1 CD3:CD28 ratio of antibodies is used for binding to the beads.

[0369] Particle-to-cell ratios of 1:500 to 500:1, and any integer values ​​therebetween, can be used to stimulate T cells or other target cells. As one of ordinary skill in the art will readily appreciate, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads may bind only a few cells, while larger beads may bind many cells. In certain aspects, cell-to-particle ratios ranging from 1:100 to 100:1, and any integer values ​​therebetween, and in other aspects, ratios encompassing 1:9 to 9:1, and any integer values ​​therebetween, 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 a preferred ratio being at least 1:1 particles / T cell. In one aspect, a particle to cell ratio of 1:1 or less is used. In a specific aspect, a preferred particle to cell ratio is 1:5. In other aspects, the particle to cell ratio can vary depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day thereafter for up to 10 days at a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In a specific aspect, the ratio of particles to cells is 1:1 on the first day of stimulation, and the ratio of particles to cells is adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, particles are added daily or every other day at a final ratio of 1:1 on the first day of stimulation and 1:5 on the third and fifth days of stimulation. In one aspect, the ratio of particles to cells is 2:1 on the first day of stimulation, and the ratio of particles to cells is adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, particles are added daily or every other day at a final ratio of 1:1 on the first day of stimulation and 1:10 on the third and fifth days of stimulation. One skilled in the art will appreciate that various other ratios may also be suitable for use with the present invention. In particular, the ratio will vary depending on the particle size and the size and type of cells. In one aspect, the most typical ratios used on the first day are approximately 1:1, 2:1, and 3:1.

[0370] In other aspects of the invention, cells such as T cells are combined with reagent-coated beads, the beads and cells are subsequently separated, and the cells are then cultured. In alternative aspects, the reagent-coated beads and cells are not separated prior to culture, but are cultured together. In other aspects, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased attachment of cell surface markers, thereby inducing cell stimulation. Preparation of CoStAR cells

[0371] Viral and non-viral genetic engineering tools can be used to produce CoStAR cells, including but not limited to T cells, NK cells, resulting in permanent or transient expression of therapeutic genes. Retroviral-based gene delivery is a mature and well-characterized technology that has been used to permanently integrate CAR into the host cell genome (Scholler J. et al., Decade-long safety and function of retroviral-modified chimeric antigen receptor Tcells. 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).

[0372] Non-viral DNA transfection methods can also be used. For example, Singh et al. describe the use of the Sleeping Beauty (SB) transposon system developed for engineering 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 are being used in clinical trials (see, e.g., ClinicalTrials.gov: NCT00968760 and NCT01653717). The same technology is applicable to engineered CoStAR cells.

[0373] A variety of SB enzymes have been used to deliver transgenes. Mátés et al. described a superactive transposase (SB100X) that is approximately 100-fold more efficient than first-generation transposases. SB100X supports 35-50% stable gene transfer in human CD34(+) cells enriched for hematopoietic stem or progenitor cells (Mátés 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 can be delivered from a polycistronic 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). cells. Proc. Natl. Acad. Sci. USA. 2016; 113: E7788-E7797) to deliver multiple transgenes. Such a system is used with the CoStAR of the present invention.

[0374] 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). Nakazawa et al. described the use of the system to generate EBV-specific cytotoxic T cells expressing HER2-specific chimeric antigen receptors (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 CD-19-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).

[0375] Transposon technology is simple and economical. A potential disadvantage is that the longer expansion protocols currently used may lead to T cell differentiation, impaired activity and poor persistence of infused cells. Monjezi et al. described the development of minicircle vectors that minimize these difficulties 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 the CoStAR of the present invention. Pharmaceutical composition

[0376] The present invention also relates to a pharmaceutical composition comprising the vector of the present invention or the cell expressing CoStAR and a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more other pharmaceutically active polypeptides and / or compounds.

[0377] 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 a nucleic acid encoding a CoStAR according to any of the above-mentioned embodiments and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided, comprising effector cells expressing the above-mentioned CoStAR and a pharmaceutically acceptable carrier. Such a formulation can be, for example, in a form suitable for intravenous infusion.

[0378] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any other component of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients preferably meet the necessary standards of toxicology and manufacturing testing and / or are included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0379] One aspect of the present invention provides a population of modified T cells expressing recombinant CoStAR. Suitable populations can be generated by the methods described above.

[0380] The modified T cell populations can be used as medicines. For example, the modified T cell populations described herein can be used in cancer immunotherapy, such as adoptive T cell therapy.

[0381] Other aspects of the present invention provide the use of the modified T cell population described herein in the preparation of a medicament for treating cancer, the modified T cell population described herein for treating cancer, and a method of treating cancer that may comprise administering the modified T cell population described herein to an individual in need thereof.

[0382] The modified T cell population can be autologous, i.e., the modified T cells are initially obtained from the same individual to whom they are subsequently administered (i.e., the donor and recipient individuals are the same). A suitable modified T cell population for administration to an individual can be generated by providing an initial population of T cells obtained from an individual, modifying the T cells to express cAMP PDE or a fragment thereof and an antigen receptor that specifically binds to a cancer cell in the individual, and culturing the modified T cells.

[0383] The T cell colony of modification can be allogenic, that is, the T cell of modification is initially obtained from the individual different from the individual applying them subsequently (i.e., donor and recipient individual are different).Donor and recipient individual can be HLA matched to avoid GVHD and other undesirable immune effects.For being administered to the T cell colony of the suitable modification of recipient individual, can be produced by including the following method: the initial colony of the T cell obtained from the donor individual is provided, the modified T cell is to express the CoStAR specifically combined with the cancer cell in the recipient individual, and the T cell of cultivation modification.

[0384] After administration of the modified T cells, the recipient individual may exhibit a T cell-mediated immune response against cancer cells in the recipient individual. This may have a beneficial effect on the individual's cancer condition.

[0385] Cancer conditions can be characterized by the abnormal proliferation of malignant cancer cells and can 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 sarcomas, 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 gall bladder and biliary tract, thyroid cancer, thymus cancer, bone cancer, and brain cancer, as well as cancer of unknown primary (CUP).

[0386] Cancer cells in an individual may be immunologically distinct from normal somatic cells in the individual (i.e., a cancerous tumor may be immunogenic). For example, a cancer cell may be able to elicit a systemic immune response in an individual against one or more antigens expressed by the cancer cell. The tumor antigen that elicits the immune response may be specific to the cancer cell or may be shared by one or more normal cells in the individual.

[0387] A subject suitable for treatment as described above can 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, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

[0388] In preferred embodiments, the subject is a human. In other preferred embodiments, non-human mammals may be used, particularly mammals routinely used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or lagomorphic). Treatment

[0389] The term "therapeutically effective amount" refers to an amount of a CoStAR or a composition comprising a CoStAR as disclosed herein that is effective to "treat" a disease or condition in an individual. In the case of cancer, a therapeutically effective amount of a CoStAR or a composition comprising a CoStAR as disclosed herein can reduce the number of cancer cells; reduce tumor size or weight; inhibit (i.e., slow down and preferably stop to some extent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. Insofar as a CoStAR or a composition comprising a CoStAR as disclosed herein can prevent the growth of existing cancer cells 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 infectious diseases (such as viral infections), a therapeutically effective amount of a CoStAR as disclosed herein or a composition comprising a CoStAR can reduce the number of cells infected by the pathogen; reduce the production or release of pathogen-derived antigens; inhibit (i.e., slow down 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 prolongs patient survival.

[0390] The cells (including T and NK cells) expressing CoStAR in the method of the present invention can be by the patient's own peripheral blood (autologous), or in the environment of hematopoietic stem cell transplantation by the peripheral blood (allogeneic) of the donor or by the peripheral blood (allogeneic) of unrelated donor in vitro generation. Alternatively, T cells or NK cells can be derived from inducible progenitor cells or embryonic progenitor cells to the in vitro differentiation of T cells or NK cells. In these cases, by one of many means (including transduction with viral vectors, with DNA or RNA transfection), DNA or RNA encoding CoStAR and optional CAR and / or TCR is introduced to produce T cells expressing CoStAR and optional CAR and / or TCR.

[0391] T cells or NK cells expressing the CoStAR of the present invention and optionally expressing a TCR and / or CAR can be used to treat hematological cancers or solid tumors.

[0392] The method for treating a disease relates to the therapeutic use of the vector or cell (including T or NK cells) of the present invention. In this regard, the vector, or T cell or NK cell can be administered to an object suffering from an existing disease or condition to alleviate, reduce or improve at least one symptom related to the disease and / or slow down, reduce or block the progress of the disease. The method of the present invention can cause or promote T cell-mediated cancer cell killing. The vector, or T cell or NK cell according to the present invention can be administered to the patient together with one or more other therapeutic agents. One or more other therapeutic agents can be co-administered to the patient. "Co-administered" means administering one or more other therapeutic agents and the vector, or T cell or NK cell of the present invention close enough in time so that the vector or T cell or NK cell can enhance the effect of one or more other therapeutic agents, or vice versa. In this regard, the vector or cell can be administered first, followed by one or more other therapeutic agents, or vice versa. Alternatively, the vector or cell and one or more other therapeutic agents can be administered simultaneously. A possible useful co-administered therapeutic agent is IL-2, because it is currently used in existing cell therapy to strengthen the activity of the cells administered. However, IL-2 therapy is associated with toxicity and tolerability issues.

[0393] As mentioned, for administration to a patient, the CoStAR effector cells can be allogeneic or autologous to the patient. In some embodiments, the allogeneic cells are further genetically modified, for example by gene editing, to minimize or prevent GVHD and / or an immune response of the patient to the CoStAR cells.

[0394] CoStAR effector cells are used to treat cancers and tumor diseases associated with the target antigen. Cancers and tumor diseases that can be treated using any of the methods described herein include tumors that are not vascularized or not yet significantly vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (such as blood tumors, such as leukemias and lymphomas) or can include solid tumors. The types of cancers 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 lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0395] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or blood-borne) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia); polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0396] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors (such as sarcomas and carcinomas) include adrenocortical carcinoma, bile duct cancer, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, 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 cancer, thyroid cancer (for example), and thyroid cancer (for example). e.g., medullary thyroid carcinoma and papillary thyroid carcinoma), pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical malignancies (e.g., cervical cancer and preinvasive cervical dysplasia), colorectal cancer, anal cancer, anal canal or anorectal cancer, vaginal cancer, vulvar cancer (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma), cancers of the head (e.g., squamous cell carcinoma), neck (e.g., squamous cell carcinoma), testicular (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma, fibroadenoma, adenomatoid tumor, and lipoma), bladder, kidney, melanoma, uterine (e.g., endometrial) cancer, urothelial (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), and ureteral carcinoma. , ureteral cancer, and bladder cancer) and CNS tumors (e.g., gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0397] When an "immunologically effective amount," "anti-tumor effective amount," "tumor suppressive effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. In general, it can be said that a pharmaceutical composition comprising T cells as described herein can be administered in an amount of 10 4 to 10 9 cells / kg body weight, in some cases 10 5 to 10 6 The T cell compositions may be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). Multiple administrations of the T cell compositions at these doses may also be performed. The cells may be administered using infusion techniques generally known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Combination therapy

[0398] The cells expressing CoStAR as described herein can be used in combination with other known agents and therapies. "Combination" used herein means delivering two (or more) different treatments to the object during the painful process of the object suffering from the disease, such as after the object has been diagnosed with the disease and before the disease is cured or eliminated or before stopping treatment for other reasons, delivering two or more treatments. In some embodiments, a treatment still exists when the second treatment is started, so that there is overlap in terms of administration. This is sometimes referred to as "simultaneous" or "parallel delivery" in this article. In other embodiments, the delivery of a treatment ends before the delivery of another treatment begins. In some embodiments of any one situation, due to combined administration, treatment is more effective. For example, the second treatment is more effective, for example, an equivalent effect is observed with less second treatment, or compared with if the second treatment is applied in the absence of the first treatment, the second treatment alleviates symptoms to a greater extent, or similar situations are observed with the first treatment. In some embodiments, delivery makes the reduction of symptoms or other parameters related to the disease greater than that observed for a treatment delivered in the absence of another. The effect of the two treatments can be partially added, completely added or greater than added. Delivery can be such that the effects of the first treatment delivered are still detectable when the second treatment is delivered.

[0399] The cells expressing CoStAR as described herein and at least one other therapeutic agent can be administered simultaneously, in the same or separate compositions or administered sequentially. For sequential administration, the cells expressing CAR as described herein can be administered first, and then other agents can be administered, or the order of administration can be reversed.

[0400] CoStAR therapy and / or other therapeutic agents, procedures or modalities may be administered during active disease or during remission or less active disease. CoStAR therapy may be administered before, concurrently with, after or during remission of other treatments.

[0401] When administered in combination, the treatment and additional agent (e.g., the second or third agent) or all can be administered in an amount or dosage higher, lower, or the same amount or dosage as the amount or dosage of each agent used alone as a monotherapy, for example. In some embodiments, the amount or dosage of CoStAR therapy, additional agent (e.g., the second or third agent) or all is lower than (e.g., at least 20%, at least 30%, at least 40% or at least 50%) the amount or dosage of each agent used alone as a monotherapy, for example. In other embodiments, the amount or dosage of CoStAR therapy, additional agent (e.g., the second or third agent) or all is lower than (e.g., at least 20%, at least 30%, at least 40% or at least 50%) the amount or dosage of each agent used alone as a monotherapy, for example, to achieve the same therapeutic effect.

[0402] In other aspects, 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 therapies), cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation, peptide vaccines (such as those described in Izumoto et al., 2008 J Neurosurg 108:963-971).

[0403] In certain instances, the compounds of the present invention are combined with other therapeutic agents, such as other anticancer agents, antiallergic agents, antinausea agents (or antiemetics), pain relievers, cytoprotective agents, and combinations thereof.

[0404] In one embodiment, the CoStAR-expressing cells described herein can be used in combination with a chemotherapeutic agent. 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, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, ofatumomab, tositumomab, brentuximab), antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclarubicin A, gliotoxin, or bortezomib), immunomodulators such as thalidomide or a thalidomide derivative (e.g., lenalidomide).

[0405] Common chemotherapeutic agents considered for combination therapy include busulfan Busulfan injection Cladribine Cyclophosphamide Cytarabine, cytosine arabinoside Cytarabine liposome injection Daunorubicin hydrochloride Daunorubicin citrate liposome injection Dexamethasone, doxorubicin hydrochloride Etoposide Fludarabine phosphate Hydroxyurea Idabit Mitoxantrone Gemtuzumab ozogamicin

[0406] In an embodiment, general chemotherapeutic agents contemplated for use in combination therapy include anastrozole Bicalutamide Bleomycin sulfate Busulfan Busulfan injection Capecitabine N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Carmustine Chlorambucil Cisplatin Cladribine Cyclophosphamide Cytarabine, cytosine arabinoside Cytarabine liposome injection Dacarbazine Dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride Daunorubicin citrate liposome injection Dexamethasone, docetaxel Doxorubicin hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide tezacitibine, gemcitabine (difluorodeoxycitidine), hydroxyurea Idabit Ifosfamide Irinotecan L-asparaginase Calcium folinate, melphalan 6-Mercaptopurine methotrexate Mitoxantrone mylotarg, paclitaxel Phoenix (yttrium 90 / MX-DTPA), pentostatin, polyphenylpropanone 20 with carmustine implant Tamoxifen citrate Teniposide 6-thioguanine, thiotepa, tirapazamine Topotecan Hydrochloride for Injection Vinblastine vincristine and vinorelbine

[0407] Treatment can be evaluated, for example, by tumor regression, reduction in tumor weight or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining efficacy of treatment can be used, including, for example, measuring response by radiographic imaging. sequence

[0408] The following sequences include the entire CoStAR and CoStAR components and are non-limiting. The components include a signal peptide (SP), a binding domain (BD), a linker, a spacer and a transmembrane domain (STM), a CD28 transmembrane fragment without extracellular or intracellular sequences (STM-CD28TM), an intracellular signaling domain (SD), and a CD40 domain and motif. Although SEQ ID NOs: 42-247 include CoStARs with N-terminal signal peptides, it is understood that the N-terminal signal peptide is removed from mature CoStARs. Furthermore, it is understood that mature CoStARs may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N-terminus (i.e., 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 range, and components may be derived from more than one source. In the event that more than one intracellular signaling domain or signaling fragment is present, the multiple domains may be in any order. It is understood that although certain proteins may contain N-terminal signal peptides when expressed, those signal peptides are cleaved and may be cleaved imprecisely when the protein is expressed, and that the resulting protein from which the signal peptide is removed contains binding domains having up to about 5 amino acid changes at the position of the N-terminal amino acid.

[0409] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

[0410] The present invention will be further illustrated in the following examples, which are given for illustrative purposes only and are not intended to limit the present invention in any way.

[0411] The embodiments of the present disclosure provided herein are described by the following exemplary numbered arrangement: 1. A chimeric co-stimulatory antigen receptor (CoStAR) comprising: an extracellular binding domain that binds to carcinoembryonic antigen (CEA), or an extracellular binding domain that binds to mesothelin (MSLN), the extracellular binding domain being operably linked to a transmembrane domain, and the first signaling domain and the intracellular domain of ICOS or a signaling fragment thereof, or the first signaling domain and the intracellular domain of NTRK1 or a signaling fragment thereof, or the first signaling domain and the intracellular domain of DAP10 or a signaling fragment thereof, or a first signaling domain and a CD40 signaling domain or a signaling fragment thereof, or A first signaling domain and one or more of a TRAF2 / TRAF3 sequence, a TRAF6 sequence, a TRAF2 sequence, or an IProx sequence. 2. The CoStAR of arrangement 1, wherein 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. 3. The CoStAR of arrangement 1, wherein the CoStAR comprises a second signaling domain. 4. The CoStAR of arrangement 3, wherein the second 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. 5. The CoStAR of arrangement 1, wherein 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. 6. The CoStAR of arrangement 2, wherein the first signaling domain comprises a full-length costimulatory domain. 7. The CoStAR according to arrangement 1, wherein the extracellular binding domain is operably linked to the transmembrane domain via a linker and / or a spacer. 8. The CoStAR of array 7, wherein the linker comprises about 5 to about 20 amino acids. 9. The CoStAR of array 7, wherein the linker comprises about 10 to about 250 amino acids. 10. The CoStAR of arrangement 1, wherein the CoStAR comprises a second extracellular binding domain. 11. The CoStAR of array 10, wherein the second extracellular binding domain comprises a ligand binding domain from CD8, CD28, or ICOS. 12. The CoStAR of arrangement 1, wherein the transmembrane domain comprises a transmembrane domain from CD28, CD8, ICOS, DAP10 or NTRK. 13. The CoStAR of arrangement 1, wherein the transmembrane domain comprises the transmembrane domain sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22. 14. The CoStAR of arrangement 1, wherein the extracellular binding domain comprises a scFv, a peptide, an antibody heavy chain variable domain, an antibody light chain variable domain, or a CEA ligand. 15. The CoStAR of any one of arrangements 1 to 14, further comprising a CD3 zeta signaling domain at the C-terminus. 16. The CoStAR of any one of arrangements 1 to 14, further comprising an N-terminal signal peptide. 17. The CoStAR according to array 16, wherein the N-terminal signal peptide comprises a signal peptide of oncostatin M (OSM), CD8α, CD2, interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF) or human IgGκ. 18. A nucleic acid encoding a CoStAR according to any one of arrays 1 to 17. 19. A vector comprising the nucleic acid of arrangement 18. 20. A cell expressing a CoStAR according to any one of arrays 1 to 16. 21. The cell of arrangement 20, wherein the cell comprises an α-β T cell, a γ-δ T cell, a regulatory T cell, a TIL, a NKT cell, or a NK cell. 22. The cell of Arrangement 20, wherein the cell co-expresses CAR or TCR. 23. A method for preparing the cell of Arrangement 20, comprising the step of transducing or transfecting the cell with the vector of Arrangement 19. 24. A method for preparing a cell population expressing a CoStAR according to any one of arrangements 1 to 16, comprising: i) detecting the expression of CoStAR on the surface of cells transfected or transduced with the vector of arrangement 19; and ii). Selecting cells identified as expressing the CoStAR. 25. A cell population enriched for cells expressing a CoStAR according to any one of arrays 1 to 16. 26. A method of treating a disease in a subject comprising the step of administering to the subject a cell according to any one of arrays 20 to 22 or a cell population according to array 25. 27. A protein comprising a consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 An amino acid sequence of a sequence wherein: a.X1 is any amino acid, b. X2 is any amino acid, c. X3 is P, X4 is any amino acid, d.X5 is Q, e.X6 is C or A, f.X7 is any amino acid, g.X8 is any amino acid, h.X9 is any amino acid, iX 10 is any amino acid, and JX 11 is any amino acid. 28. The protein of array 27, wherein said protein is part of a TRAF domain. 29. The protein of any one of the preceding arrangements, wherein the protein is part of a TRAF2 domain. 30. A protein according to any one of the preceding arrangements, wherein: a. The sequence comprises: i.X1 is S or P, ii. X2 is V, H or Y, iii.X4 is I, Q, V, iv.X7 is T or S, v.X8 is D, vi.X9 is K, D or G, vii.X 10 is T, S, G, or A, and viii.X 11 is D, S, N or D; or b. The sequence is at least 60% identical to a). 31. A protein comprising a consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 An amino acid sequence of a sequence wherein: a.X1 is any amino acid, b. X2 is any amino acid, c. X3 is P, S, A or T, d.X4 is any amino acid, e.X5 is Q or E, f.X6 is E, g.X7 is any amino acid, h.X8 is any amino acid, i.X9 is any amino acid, JX 10 is any amino acid, and kX 11 is any amino acid. 32. The protein of array 31, wherein said protein is part of a TRAF domain. 33. The protein of any one of the preceding arrangements, wherein the protein is part of a TRAF2 domain. 34. A protein according to any one of the preceding arrangements, wherein: a. The sequence comprises: i.X1 is P, A, M, T, S, R, V, H, ii. X2 is F, A, L, I, T, V, G, C, A, F, P, iii.X4 is K, V, I, H, A, Q, T, E, S, iv.X7 is C, T, E, D, S, A, v.X8 is A, L, G, Y, I, Q, D, E, vi.X9 is F, H, K, R, P, A, G, Y, E, N, vii.X 10 is R, G, E, K, A, S, D, C, C, P, V, and viii.X 11 is S, C, D, P, W, T, A, G, E; or b. The sequence is at least 60% identical to a). 35. The protein of any one of the preceding arrangements, wherein the sequence is a portion of a Traf2 / 3 sequence. 36. A protein comprising an amino acid sequence comprising a consensus C or XXXX, wherein: a.X1 is P, b. X2 is any amino acid, c.X3 is E, d.X4 is any amino acid, e. X5 is any amino acid, and f.X6 is Ac / Ar. 37. The protein of array 36, wherein said protein is part of a TRAF domain. 38. The protein of any one of the preceding arrangements, wherein the protein is part of a TRAF6 domain. 39. A protein according to any one of the preceding arrangements, wherein a. The sequence comprises: i.X2 is Q, P, E, V, T or S, ii.X4 is I, L, M, N, S, T, N, D, iii. X5 is N, D, R, S, G or Y; or b. The sequence is at least 60% identical to a). 40. An amino acid sequence comprising: a) one or more of 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). 41. A TRAF domain in a CD40 protein, wherein the TRAF domain comprises one or more of the following: 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). 42. A TRAF6 domain sequence comprising: PQEINF, PEEMSW, PPENYE or PQENSY. 43. A TRAF2 / 3 domain sequence comprising: PVQET, TQEET, SKEET, AVEET or PVQET. 44. A TRAF2 domain sequence comprising: SVQE, AVEE or PEEE. 45. A TRAF 6 or TRAF 2 / 3 domain comprising: a) the amino acid sequence of one of 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) an amino acid sequence that is at least 70% identical to a); or c) an amino acid sequence at least 80% similar to a). 46. ​​The protein, amino acid sequence or TRAF domain of any one of the preceding arrangements, wherein the sequence is within the CD40 protein sequence. 47. A protein, amino acid sequence or TRAF domain as described in any of the preceding arrangements, wherein the sequence is within a CD40 protein and is located at the corresponding TRAF2, TRAF2 / 3 and / or TRAF6 domain within the CD40 protein. 48.TRAF2 / TRAF3 domain containing consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of a.X1 is any amino acid, b. X2 is any amino acid, c. X3 is P, X4 is any amino acid, d.X5 is Q, e.X6 is C or A, f.X7 is any amino acid, g.X8 is any amino acid, h.X9 is any amino acid, iX 10 is any amino acid, and JX 11 is any amino acid. 49.TRAF2 / TRAF3 domain containing consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of a.X1 is any amino acid, b. X2 is any amino acid, c. X3 is P, S, A or T, d.X4 is any amino acid, e.X5 is Q or E, f.X6 is E, g.X7 is any amino acid, h.X8 is any amino acid, i.X9 is any amino acid, JX 10 is any amino acid, and kX 11 is any amino acid. 50. TRAF6 domain comprising a consensus amino acid sequence of C or X1X2X3X4X5X6, wherein a.X1 is P, b. X2 is any amino acid, c.X3 is E, d.X4 is any amino acid, e. X5 is any amino acid, and f.X6 is Ac / Ar. 51. A CD40 domain comprising one or more of the following amino acid sequences: a. TRAF2 / TRAF3 domain, which contains consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of i.X1 is any amino acid, ii. X2 is any amino acid, iii. X3 is P, X4 is any amino acid, iv.X5 is Q, v.X6 is C or A, vi. X7 is any amino acid, vii. X8 is any amino acid, viii. X9 is any amino acid, ix.X 10 is any amino acid, and xX 11 is any amino acid. b. TRAF2 / TRAF3 domain, which contains consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of i.X1 is any amino acid, ii. X2 is any amino acid, iii. X3 is P, S, A or T, iv. X4 is any amino acid, v.X5 is Q or E, vi.X6 is E, vii. X7 is any amino acid, viii. X8 is any amino acid, ix.X9 is any amino acid, xX 10 is any amino acid, and xi.X 11 is any amino acid. c. TRAF 6 domain comprising a consensus amino acid sequence of C or X1X2X3X4X5X6, wherein i.X1 is P, ii. X2 is any amino acid, iii.X3 is E, iv. X4 is any amino acid, v. X5 is any amino acid, and vi.X6 is Ac / Ar. 52. A chimeric co-stimulatory antigen receptor (CoStAR) comprising: an extracellular binding domain that binds to carcinoembryonic antigen (CEA) or an extracellular binding domain that binds to mesothelin (MSLN); a transmembrane domain connected 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 sequence of a variant TRAF2 / TRAF3 sequence or a variant TRAF6 sequence or a variant TRAF2 sequence, wherein the variant CD40 does not comprise SEQ ID NO: 42. 53. The CoStAR of array 52, wherein the first signaling domain comprises a signaling domain or signaling fragment of CD28 or CD278 (ICOS). 54. The CoStAR of arrangement 52, wherein the first signaling domain comprises a full-length costimulatory domain. 55. The CoStAR of array 52, wherein the extracellular binding domain is operably linked to the transmembrane domain via a linker and / or a spacer. 56. The CoStAR of arrangement 55, wherein the linker comprises about 5 to about 20 amino acids. 57. The CoStAR of arrangement 55, wherein the linker comprises about 10 to about 250 amino acids. 58. The CoStAR of arrangement 55, wherein the transmembrane domain comprises a transmembrane domain from CD28, CD8, ICOS, DAP10, or NTRK. 59. The CoStAR of arrangement 55, wherein the transmembrane domain comprises the transmembrane domain sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22. 60. The CoStAR of array 55, wherein the extracellular binding domain comprises a scFv, a peptide, an antibody heavy chain variable domain, an antibody light chain variable domain, or a CEA ligand. 61. A nucleic acid encoding a CoStAR according to any one of sequences 27 to 60. 62. A vector comprising the nucleic acid of arrangement 61. 63. A cell expressing the CoStAR of any one of arrays 27 to 62. 64. The cell of arrangement 63, wherein the cell comprises an α-β T cell, a γ-δ T cell, a regulatory T cell, a TIL, a NKT cell, or a NK cell. 65. The cells of arrangement 64, wherein the cells co-express CAR or TCR. 66. A method for preparing a cell according to any one of arrays 63 to 65, comprising the step of transducing or transfecting the cell with the vector according to array 62. 67. A method for preparing a cell population expressing a CoStAR according to any one of arrangements 52 to 60, comprising: a. detecting the expression of CoStAR on the cell surface transfected or transduced with the vector of arrangement 62; and b. Select cells identified as expressing the CoStAR. 68. A cell population enriched for cellular expression of a CoStAR according to any one of arrays 52 to 60. 69. A method of treating a disease in a subject comprising the step of administering to the subject a cell according to any one of arrays 63 to 65 or a cell population according to array 68. 70. A protein comprising the amino acid sequence of one of SEQ ID NOs: 510-519. 71. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 510. 72. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 511. 73. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 512. 74. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 513. 75. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 514. 76. The protein of array 70, wherein said protein comprises the sequence of SEQ ID NO: 515. 77. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 516. 78. The protein of array 70, wherein said protein comprises the sequence of SEQ ID NO: 517. 79. The protein of array 70, wherein said protein comprises the sequence of SEQ ID NO: 518. 80. The protein of arrangement 70, wherein said protein comprises the sequence of SEQ ID NO: 519. Example Example 1 - Generation of T cells expressing CoStAR

[0412] Materials and methods

[0413] Construct design - MFE23 CoStAR consists of a single-chain antibody fragment nucleotide sequence derived from CEA-specific MFE23, humanized (hu) MFE23, CEA6, BW431 / 26, or huT84.66, and an oncostatin M1, CD8α, CD2, IL-2, GM-CSF, or hIgGγVIII leader sequence. Each CoStAR has an extracellular spacer domain derived from CD8 or CD28 or a truncated CD28, as well as a signaling domain derived from CD28 and CD40. The construct was cloned into pSF.Lenti (Oxford Genetics) containing the MND promoter and separated from the truncated CD34 marker gene via the P2A cleavage sequence.

[0414] Lentiviral production—Lentiviral production was performed using a three-plasmid packaging system (CellBiolabs, San Diego, USA) by mixing 10 μg of each plasmid plus 10 μg of the pSF.Lenti lentiviral plasmid containing the transgene in serum-free RPMI containing 50 mM CaCl2. The mixture was added dropwise to a 75 cm 2In 50% confluent 293T cell monolayers in flasks. Viral supernatants were collected 48h and 72h after transfection, combined and concentrated using LentiPac lentiviral supernatant concentrate (GeneCopoeia, Rockville, Maryland, USA) according to the manufacturer's instructions. The lentiviral supernatant was concentrated 10 times and used to directly infect primary human T cells in the presence of 4 μg / ml polybrene (Sigma-Aldrich, Dorset, UK). Before adding lentiviral supernatant, peripheral blood mononuclear cells were isolated from normal healthy donors before activation for 24 hours with T cell activation and amplification beads (Invitrogen) according to the manufacturer's instructions.

[0415] 96 hours after infection, cell transduction was assessed using CEA.hFc protein and an anti-hFc-PE secondary antibody plus anti-CD34-APC or anti-CD34-PE antibody alone. Cells were then further expanded using x10 donor-mismatched irradiated PBMC feeder cells at a ratio of 1:20-1:200 in RPMI + 10% FCS supplemented with 1 μg / ml PHA and 200 IU / ml IL-2. After 14 days, cells were stained as described above and stored until assayed.

[0416] Functional assays were performed by mixing CoStAR-positive or -negative cells with wild-type or OKT3-engineered CEA-positive LoVo or LS174T cells. Briefly, T cells were mixed with LoVo cells in 96-well plates at different ratios, and IFNγ or IL-2 was measured by ELISA. The remaining cells were incubated with a 1:10 dilution of WST-1 reagent (Sigma, UK) for 30 min before reading the absorbance at 450 nm. Cytotoxicity % was determined using the following equation = 100-((experimental reading - T-cell alone) / (tumor alone))×100.

[0417] First, incubate the cells at 37°C with 1 × 10 7 T cells were loaded with 10 μM eFluor 450 proliferation dye (eBioscience, UK) at a concentration of 10 cells / ml for 10 minutes, and then the cells were incubated on ice in 5 volumes of cold T cell culture medium for 5 minutes for proliferation assay. The cells were then washed excessively to remove unbound dye and added to co-cultures containing tumor cells. On days 2, 6, and 10, cells were removed, a 1:200 dilution of DRAQ7 was added, and cells were analyzed using a MACSQuant cell counter and MACSQuantify software.

[0418] Cell counts for proliferation assays were performed by removing cells from the wells and staining with anti-CD2 PerCP eFluor710 antibody (eBioscience, UK) for 20 minutes in the dark, followed by DRAQ7 staining and counting using a MACSQuant analyzer.

[0419] result

[0420] Primary human T cells were isolated from buffy coats obtained from NHSBT. T cells were isolated by Ficoll-mediated separation and a T cell negative isolation kit (StemCell Technologies). The isolated T cells were activated with human T cell activation and amplification beads (Invitrogen, UK). The cells were incubated with concentrated lentiviral particles and amplified for several days. The lentivirus contains the DNA sequence of the MFE.CoStAR.2A.tCD34 construct (MFE23.scFv fused to full-length human CD28 co-expressed with truncated human CD34 via a 2A cleavage sequence). As outlined in the Materials and Methods, successfully transduced cells were further amplified using irradiated feeder cells. Donor 1 transduction was measured to be 22.69% (17.15CD34+ / CoStAR+ plus 5.53%CD34- / CoStAR+), donor 2 was measured to be 20.73%, and donor 3 was 13.34%. CoStAR-expressing cells were enriched using anti-CD34 antibody, and a T cell population with greater than 90% CoStAR-positive cells was obtained.

[0421] To generate a physiologically relevant in vitro model to test the effects of CoStAR on T cell activity, non-transduced and transduced cells were tested against the CEA+ tumor cell lines LoVo and LS174T. To enable activation of T cells in response to mismatched tumor lines, we engineered tumor cells to express an anti-CD3 single-chain antibody fragment anchored to the cell membrane via a synthetic transmembrane domain and genetically isolated from a GFP marker using an IRES element to visualize transduced cells using flow cytometry.

[0422] Single cell clones of LoVo and LS174T were generated from a large number of transfectants. Non-transduced and CoStAR-transduced T cells were mixed with wild-type non-transduced or OKT3-engineered LS174T or LoVo cells at different effector-to-target ratios. After 24 hours, the co-culture medium was removed for IL-2 ELISA measurement. Activation-dependent IL-2 secretion in response to OKT3-engineered LS174T cells was observed in CoStAR+ and CoStAR- T cell populations from three donors, and only background IL-2 secretion in response to non-engineered tumor cells was observed from transduced and non-transduced T cells ( Figure 3ACoStAR-enhanced IL-2 secretion was observed in all three donors tested for OKT3 engineered tumor cells. This effect was most pronounced at E:T ratios of 8:1 and 16:1, while at higher E:T ratios, IL-2 secretion was too low to be accurately measured. At lower effector-to-target ratios, IL-2 secretion by non-transduced cells appears to saturate. These observations were replicated in LoVo cells, with similar results observed for two of the three donors tested for LS174T ( Figure 3D and E).

[0423] To determine the effect of CoStAR on T cell expansion, transduced or non-transduced T cells were mixed with wild-type or OKT3-GFP engineered LoVo cells, and the total cell number was counted 3 days later. Figure 4A ) and the absence of IL-2 ( Figure 4B ), CoStAR enhanced the survival and / or proliferation of engineered T cells in response to LoVo-OKT3 but not wild-type LoVo cells. To further investigate this phenomenon, cell proliferation assays were performed in T cells from two donors using proliferation dyes to count the number of cell cycles per population over 6 days ( Figure 4C (A and D) Compared to non-engineered cells in response to LoVo-OKT3, a greater proportion of CoStAR-engineered cells underwent 5, 6, or 7 proliferation cycles in 6 days, whereas CoStAR-transduced and non-transduced cells underwent an average of approximately 2 cycles in the same duration in response to wild-type LoVo.

[0424] A variety of fusion receptors consisting of CD28 fused to an additional costimulatory domain at the N-terminus were generated. The costimulatory domains obtained from CD137, CD2, CD29, CD134, CD150, CD40, GITR and the signaling domain from the IL-2 receptor gamma chain (IL2Rγ) were selected. Receptors close to the receptors used in previous inducible costimulatory studies were included. This receptor, called CD28 (IEV), was truncated so that the C-terminal motif of CD28 was the amino acid triplet 'IEV'. The sequence was generated de novo by Genewiz and cloned into a lentiviral vector under the EF1α promoter together with the CD34 marker gene separated from the fusion CoStAR by a 2A self-cleaving peptide. Primary CD8+ T cells were separated using EasySep beads (StemCell Technologies) and activated with anti-CD3 / anti-CD28 activation / amplification Dynabeads before adding lentiviral particles. After a short expansion period, cells were mixed with LoVo or LoVo-OKT3 cells, which included anti-CD107a antibodies as well as brefeldin and monensin, and after a 16-hour incubation, fixed and stained with antibodies to a marker gene (CD34) and antibodies to IL-2, IFNγ, and bcl-xL. Analysis was performed using a MACSQuant analyzer and MACSQuantify software. Figure 5 IL-2 responses from CD34- (non-CoStAR transduced) and CD34+ (CoStAR transduced) are shown. Statistical analysis shows that when possessing variant CoStAR receptors, the proportion of cells that induce IL-2 production by all receptors tested is significantly increased. Three other readings were measured simultaneously: IFNγ, a cytokine released under normal signal 1 conditions but enhanced by co-stimulation; CD107a, a marker of degranulation; and bcl-xL, an anti-apoptotic protein upregulated by co-stimulation. The involvement of CoStAR enhances all effector functions analyzed to varying degrees. CD28.CD2 and CD28.CD40 fusion receptors appear to trigger the most robust responses of all tested receptors (see Figure 6). Example 2

[0425] The effects of CD28-based and CD28.CD40-based CoStARs on cytokine secretion-based populations were compared. Primary T cells from three donors were transduced with CD28 (IEV)-truncated CoStAR, full-length CD28 CoStAR, or CD28.CD40 CoStAR (having full-length CD28 as shown in SEQ ID NO: 439, but lacking the N-terminal N and K residues), or were not transduced. CoStAR-expressing T cells were enriched using the CD34 marker gene, and the expanded cells were then mixed with LoVo-OKT3 cells and analyzed for IL-2 secretion by ELISA (see Figure 7 Non-transduced cells produced an average of 0.80 ng / ml IL-2, while CD28(IEV) and full-length CD28 CoStAR produced 4.6 ng / ml and 5.0 ng / ml IL-2, respectively. However, CD28.CD40 induced an average of 29.0 ng / ml IL-2 in three donors, demonstrating a clear benefit of incorporating CD40 into the basic CD28-based CoStAR.

[0426] Next, the effect of CoStAR on T cell expansion was analyzed. T cells from 7 donors were transduced with CD28 CoStAR or CD28.CD40 CoStAR with anti-CA125 (196-14) or anti-folate receptor (MOV-19) scFv or anti-folate receptor peptide (C7) antigen binding domains. Additional cells were transduced with CD28 CoStAR with anti-CEA scFv as mismatch controls. The cells were then mixed with the CA125+ / folate receptor+ / CEA- cell line OvCAR3 (OvCAR-OKT3) engineered to express membrane-bound OKT3. T cell counts were performed after 7 days, 14 days, and 21 days, and fresh OvCAR-OKT3 was added on the 7th and 14th days. Limited expansion was observed for cells with anti-CA125 scFv (mean fold expansion: CD28: 15.1; CD28.CD40: 69.1), however, cells targeting the folate receptor with scFv did expand in both the CD28 and CD28.CD40 cohorts (mean fold expansion: CD28: 186.7; CD28.CD40: 1295.0). When the C7 peptide was used to target the folate receptor, more limited expansion was observed (mean fold expansion: CD28: 71.5; CD28.CD40: 28.0). The control CEA-targeted receptor showed limited expansion (mean fold expansion: 28.0).

[0427] To better understand the synergistic effects of signal 1 and signal 2, T cells were engineered with a mouse constant domain-modified TCR that recognizes the CEA peptide (691-699) in the context of HLA-A*02 and CD28 or CD28.CD40 CoStAR targeting the cell surface CEA protein. As a control, cells were also transduced with a CA125-specific CD28 CoStAR. T cells were mixed with HLA-A*02+ / CEA+ H508 cells and analyzed for cytokine production by intracellular flow cytometry staining. Flow cytometry was gated using antibodies against the mouse TCRβ constant domain (labeling TCR-engineered cells) and the DYKDDDDK (SEQ ID NO: 449) epitope tag (labeling CoStAR-engineered cells). Thus, TCR- / CoStAR-, TCR+ / CoStAR-, TCR- / CoStAR+, and TCR+ / CoStAR+ cells in each co-culture well could be analyzed. The cytokine production of each subset of CD4+ or CD8+ T cells was then plotted ( Figure 9 ). In CD4+ cells, CD28.CD40 CoStAR enhanced the production of CD137 and TNFα, which was higher than that of TCR stimulation alone. However, due to the dependence of TCR on CD8, the TCR response in CD4+ cells was poor. In CD8+ cells, IL-2 and CD107a had more robust effector activity, especially showing stronger induction in the CD28.CD40 CoStAR group. In order to better compare the receptors, only the effector activity of the TCR+ / CoStAR+ group in CD4+ and CD8+ cells was plotted (Figure 10). In CD4+ cells, CD28.CD40 significantly enhanced the induction of CD137 compared to CEA or mismatched targeting CD28 CoStAR. In CD8+ cells, CD137 induction was significantly increased compared to CEA or mismatched targeting CD28 CoStAR, however, CD107a induction was increased compared to control CoStAR. Therefore, CD28.CD40 showed enhanced effector activity in a wide range of models and effector activities. Example 3

[0428] To evaluate the co-stimulation of CD40-carrying CoStARs, primary human T cells were mock transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 CoStARs, each with a CD34 marker gene separated by a 2A cleavage peptide. MFE23 is a single-chain Fv antibody with high affinity for carcinoembryonic antigen (CEA). After in vitro culture, MACS was used to TMCells were enriched for CD34 using paramagnetic selection reagent (Miltenyi Biotech) and then expanded using irradiated feeder cells. + T cell expansion, and MFE23.CD28.CD40CoStAR further enhanced expansion ( Figure 11 ).

[0429] To evaluate co-stimulatory activity and persistence, T cells mock-transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 were co-cultured with LoVo-OKT3 cells at an effector-to-target ratio of 8:1 in the presence (200 IU / ml) or absence of exogenous IL-2. On days 1, 4, 7, 11, and 18, cells were harvested and the number of viable T cells was counted on a MACSQuant flow cytometer using anti-CD2 reagent. In the absence of tumor stimulation and IL-2, the cell number decreased, as expected ( Figure 12A In the absence of stimulation but in the presence of IL-2, there was more pronounced cell survival but no specific growth ( Figure 12B In the presence of tumor but without IL-2, mock cells showed no specific survival. MFE23.CD28 CoStAR mediated a significant expansion doubling within the first 4 days, which then declined. MFE23.CD28.CD40 mediated a greater expansion until day 7, which then steadily declined ( Figure 12C Under identical conditions but in the presence of IL-2, mock- and MFE23.CD28-transduced cells exhibited a 20-fold expansion over 18 days, while MFE23.CD28.CD40 cells expanded more than 60-fold ( Figure 12D Thus, CD28.CD40-based receptors exhibited superior expansion and survival both in the presence and absence of exogenous IL-2 stimulation.

[0430] T cells mock-transduced and transduced with MFE23.CD28 or MFE23.CD28.CD40 CoStAR were then tested for cytokine production. Supernatants obtained from T cell / tumor co-cultures were subjected to bead array analysis. Engineered T cells were incubated with LoVo-OKT3 cells at a 1:1 effector to target ratio for 24 hours and supernatants were collected. Conditioned supernatants were also collected from the same number of T cells alone or LoVo-OKT3 cells alone. TMHuman TH1 / TH2 cytokine panel (Biolegend) analyzes the production of IL-2, IFN-γ, TNFα, IL-4, IL-5, IL-13, IL-17A, IL-17F, IL-22, IL-6, IL-10, IL-9, and IL-21 ( Figures 13A-13M ). Cytokines are very low or undetectable in culture media from T cells or tumors alone. However, when co-cultured with tumors, cytokine production is enhanced. MFE23.CD28 enhanced the production of IL-2, IL-5, IL-17A / 17F, IL-10, IL-9, and IL-21 compared to the simulation. However, MFE23.CD28.CD40 also enhanced the production of TNFα, IL-13, and IL-22. MFE23.CD28.CD40 also enhanced the production of a large number of cytokines that were greater than the number of cytokines elicited by MFE23.CD28 (IL-2, IL-9, and IL-17F), and also reduced the production of some cytokines to below the levels seen with MFE23.CD28 (IL-5 and IL-10). Together, these data demonstrate that the addition of CD40 to CD28-based co-stimulatory receptors enhances and / or modulates their specific activity for chemokine production.

[0431] T cells that were mock-transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40 were further tested for chemokine production. TMThe human proinflammatory chemokine panel analyzes the production of IL-8 (CXCL8), IP-10 (CSCL10), Eotaxin (CCL11), TARC (CCL17), MCP-1 (CCL2), RANTES (CCL5), MIP-1a (CCL3), MIG (CXCL9), ENA-78 (CXCL5), MIP-3α (CCL20), GROα (CXCL1), I-TAC (CXCL11), and MEP-1β (CCL4). Chemokines are very low or undetectable in culture medium from T cells alone. However, when co-cultured with tumors, chemokine production is enhanced. Compared to mock, MFE23.CD28 enhanced the production of CXCL5, CXCL10, CXCL11, CCL17, and CCL20. However, MFE23.CD28.CD40 also enhanced the production of CCL2, CXCL1, and CXCL9. MFE23.CD28.CD40 also further enhanced the production of certain cytokines to greater amounts than those elicited by MFE23.CD28 (CXCL1, CXCL9, CXCL10, CXCL11, CCL17, CCL2, CXCL9, CCL5, and CCL20), while reducing the production of some cytokines to below the levels seen with MFE23.CD28 (CCL4). Together, these data demonstrate that the addition of CD40 to CD28-based costimulatory receptors enhances and / or modulates their specific activity for chemokine production.

[0432] Test the functional activity of CoStAR against cancer targets. With CD28 or CD28.CD40 CoStAR transduction cells engineered with scFv binding domains specific for FolR or CA125 (scFv MOV19 and scFv 196-14, respectively). Human folate receptor alpha (FolR) represents a suitable target for a variety of tumors (including ovary, head and neck, kidney and lung), while CA125 represents an alternative target for ovarian cancer. Primary human T cells from 6 healthy donors were engineered with 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28 or MOV19.CD28.CD40 receptors, all of which have DYKDDDDK epitope tags for detection. Before using intracellular staining to analyze effector activity in epitope tag positive and negative populations, transduced cells were mixed with FolR+ / CA125+OvCAR-OKT3 cells. Compared with mock-transduced cells, CD28 and CD28.CD40 engineered cells were observed to express IL-2 ( Figure 15A and 15B ), TNFα( Figure 15C and 15D)、CD137( Figure 15E and 15F ) and BCL-xL( Figure 15G and 15H ), but specific BCL-xL induction by MOV19.CD28 was less pronounced compared to MOV19.CD28.CD40.

[0433] The expansion of mock-transduced TILs or TILs engineered with MOV19.CD28.CD40 CoStAR and stimulation of CD137 production by patient-matched tumor digests was evaluated ( FIG16 ). Three donor tumors were tested that showed varying levels of FolR on digests, ranging from negative ( Figure 6A ), low expression ( Figure 6B ) to high expression ( Figure 6C CoStAR-negative TILs in mock- and FolR-negative digest-matched CoStAR-engineered TIL populations exhibited similar levels of CD137 upregulation following tumor co-culture, which was not enhanced by the presence of CoStAR ( Figure 16D In TILs exposed to digests low in FolR expression, activity was enhanced in CoStAR+ cells compared to CoStAR-, with CD137 expression increasing from <10% to >20% ( Figure 16E In TILs exposed to FolR-high tumor digests, activity increased from approximately 20% in the CoStAR- population to approximately 50% in the CoStAR+ population ( Figure 16F ).

[0434] FolR targeting of CoStAR was examined for enhancement of effector function. In response to FolR+ tumor digests, MOV19.CD28.CD40 enhanced CD137 expression from ˜20% to ˜50% ( Figure 17A ), increased TNFα production from 10% to 15% ( Figure 17B ), and enhancing IL-2 production from 2% to 5% ( Figure 17C ).

[0435] CoStAR-mediated stimulation with soluble ligands was also examined. T cells from three healthy donors were engineered with either MOV19.CD28 or MOV19.CD28.CD40 CoStAR and activated with fixed OKT3 to provide stimulation in the absence of FolR, or with OvCAR-OKT3 to provide both TCR and CoStAR activity. Following OKT3 stimulation, Bcl-XL activity increased by 10% to 20% in all three donors ( Figure 18A ), while IL-2 increased from 0% to 12% ( Figure 18B), TNFα increased from 0% to 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 ∼20% in CD28CoStAR and ∼35% in CD28.CD40CoStAR ( Figure 18D ), IL-2 induction was enhanced by ∼20% in CD28 CoStAR and 30–50% in CD28.CD40 CoStAR ( Figure 18E ), and TNFα production was enhanced by 20-30% in CD28 CoStAR and 25-50% in CD28.CD40 CoStAR ( Figure 18F Exogenous soluble FolR had no inhibitory effect on any of these effector functions. Example 4

[0436] Materials and methods

[0437] Construct design - MFE23, MOV19 and 196-14 CoStAR constructs include single-chain antibody fragment nucleotide sequences derived from MFE23 (CEA specific), MOV19 (folate receptor α specific) or 196-14 (CA125 specific), in which the oncostatin M1 leader sequence is fused to the costimulatory domain. The costimulatory domain contains an extracellular spacer and transmembrane domain derived from human CD8 or CD28 and a signaling domain of CD28, CD2 or CD137 and / or wild-type or mutant CD40 variants. Some CoStARs described in detail herein include the human PD1 extracellular domain fused to CD28 and CD40. The receptor is cloned with a P2A cleavage sequence and a truncated form of human CD34 to allow detection of transduced cells. The CoStAR nucleotide sequence is codon-optimized and synthesized by Genewiz Inc. The construct is cloned into a third-generation lentiviral vector.

[0438] Prior to the addition of lentiviral supernatant, peripheral blood mononuclear cells were isolated from normal healthy donors prior to activation with T cell activation and expansion beads (Invitrogen) for 24 hours according to the manufacturer's instructions.

[0439] 96 hours after infection, cell transduction was assessed using CEA.hFc protein (R&D Systems) and an anti-hFc-PE secondary antibody plus anti-CD34-APC or anti-CD34-PE antibody alone. Cells were then further expanded using x10 donor-mismatched irradiated PBMC feeder cells at a ratio of 1:20 to 1:200 in RPMI + 10% FCS supplemented with 30 μg / ml OKT3 and 200 IU / ml IL-2. After 14 days, cells were stained as described above and stored until assayed.

[0440] Functional assays were performed by mixing CoStAR positive or negative cells with wild type or OKT3 engineered CEA positive LoVo cells. Briefly, T cells were mixed with LoVo cells at different ratios in 96-well plates. For flow analysis, co-cultures were incubated with brefeldin, monensin and anti-CD107a antibodies for 16 h, and then the cells were stained with Fixable Viability Dye ef450 (eBiosciences), fixed with 4% paraformaldehyde, and then permeabilized with Fix / Perm wash buffer (BD Biosciences). The cells were then stained with anti-CD34 or anti-DYKDDDDK antibodies to distinguish CoStAR+ and CoStAR- populations, anti-IL-2, anti-TNFα and anti-IFNγ antibodies (Biolegend). For soluble analyte analysis, supernatants were collected and analyzed by ELISA, cytokine bead array (LEGENDPLEX TM Human Th cytokine Panel (12-plex) or chemokine bead array (LEGENDPLEX TM Human pro-inflammatory chemokine panel (13-plex)) was used for analysis.

[0441] Proliferation assays were performed by mixing T cells and tumor cells at an effector to target ratio of 8:1 in complete T cell medium (TCM:RPMI supplemented with 10% FCS, 0.01M HEPES and 1% penicillin / streptomycin, 50mM β-mercaptoethanol) in the presence or absence of IL-2. Cell counts were performed at the indicated time points, and fresh tumor cells were added in restimulation assays for a final E:T of 8:1. Cell counts for proliferation assays were performed by removing cells from the wells and staining with anti-CD2 PerCPeFluor710 antibody (eBioscience, UK) for 20 min in the dark, followed by DRAQ7 staining and counting using a MACSQuant analyzer. Example 5

[0442] To determine which receptor signaling motifs contribute to CD40-enhanced CoStAR activity, variant receptors harboring mutations in the TRAF6 binding motif (MFE23.CD28.CD40 (PQEINF mutated to AQAINF)), the TRAF2 binding motif (MFE23.CD28.CD40 (SVQE mutated to AVQA)), or the TRAF1 / 2 / 3 / 5 binding motif (MFE23.CD28.CD40 (PVQET mutated to AVAEA)) were generated. Primary human T cells isolated from three separate healthy donors were transduced with the indicated CoStARs and enriched for CoStAR expression via CD34. Co-cultures were established with LoVo-OKT3 cells, and supernatants were collected for cytokine analysis.

[0443] IL2 production was very low in non-transduced cells, but was elevated in cells expressing wild-type MFE23.CD28.CD40CoStAR. IL2 production was minimally affected by mutations in SVQE, PQEINF, or Q263A. However, mutations in the PVQET motif significantly affected IL2 production by T cells.

[0444] Simultaneously, the proliferation of engineered T cells in response to LoVo-OKT3 cells was assessed after two rounds of stimulation. Non-transduced cells failed to survive even after a single round of stimulation, while cells expressing the wild-type receptor underwent a 10-fold expansion after two rounds of stimulation over 14 days. Cells harboring either the SVQE-AVQA mutation or the PQEINF-AQAINF mutation showed a reduced ability to undergo proliferation, which was further exacerbated in cells harboring the Q263A mutation. However, cells harboring the PVQET-AVAEA mutation in the TRAF2 / 3 binding region exhibited a complete inability to proliferate after two rounds of stimulation. Example 6

[0445] CoStARs designed to bind to CEA consist of single-chain antibody fragment nucleotide sequences derived from CEA-specific MFE23, humanized (hu) MFE23, CEA6, BW431 / 26, or huT84.66, and oncostatin M1, CD8α, CD2, IL-2, GM-CSF, or hIgGγVIII leader sequences. Each CoStAR has an extracellular spacer domain derived from CD8, CD28, or truncated CD28, as well as signaling domains derived from CD28 and CD40. The construct was cloned into pSF.Lenti (Oxford Genetics) containing the MND promoter and separated from the truncated CD34 marker gene via the P2A cleavage sequence.

[0446] Lentiviral production—Lentiviral production was performed using a three-plasmid packaging system (CellBiolabs, San Diego, USA) by mixing 10 μg of each plasmid plus 10 μg of the pSF.Lenti lentiviral plasmid containing the transgene in serum-free RPMI containing 50 mM CaCl2. The mixture was added dropwise to a 75 cm 2 The 293T cells were isolated from healthy donors at a concentration of 50% in a flask. Viral supernatants were collected 48 h and 72 h after transfection, combined and concentrated using Lenti-X lentiviral supernatant concentrate (Takara Bio Inc. Japan) according to the manufacturer's instructions. The lentiviral supernatant was concentrated 10 times and directly infected with primary human T cells at an MOI of 3-5 in the presence of 4 μg / ml polybrene (Sigma-Aldrich, Dorset, UK). Peripheral blood mononuclear cells were isolated from normal healthy donors before activating with T cell activation and amplification beads (Invitrogen) for 24 hours according to the manufacturer's instructions before adding lentiviral supernatant.

[0447] 96 hours after infection, cell transduction was assessed using CEA.hFc protein and anti-hFc-PE secondary antibody plus anti-CD34-APC or anti-CD34-PE antibody alone. Cells were then further expanded at a 1:200 ratio using x10 donor-mismatched irradiated PBMC feeder cells in T cell culture medium (RPMI 1640, 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, and 50 u / ml penicillin / streptomycin), 200 IU / ml IL-2, and a final concentration of 30 ng / ml anti-CD3 (OKT3). After 12-14 days, cells were stained as described above and stored for assay.

[0448] Functional assays were performed by mixing CoStAR-positive or -negative cells with wild-type or OKT3-engineered CEA-positive cell lines (LoVo, HT29, SW480, H508). Briefly, T cells were mixed with target cells at varying ratios in 96-well plates, and cytokine release was measured by ELISA and MSD analysis.

[0449] Cytotoxicity assay was performed using an xCELLigence RTCA SP real-time cell analyzer system (Acea). A program was generated to test the pore conductivity (cell index) of a 96-well PET E plate for every 15 min during the experiment (until 250 hr). 50 μL of cell culture medium was added to the wells of the test cell index and incubated for 30 minutes at room temperature (RT). The E plate was added to the RTCASP device and background conductivity readings were measured.

[0450] The optimal density of target cells for inoculation is defined as the density that reaches a stable cell index 24 to 36 hours after the start of the assay and does not decrease without intervention (i.e., addition of Triton-x-100 or effector cell population) before the end of the assay. Target cells at the optimal density for killing assays (cell line dependent) are counted using a quantitative method capable of distinguishing dead cells. The E plate is removed from the device, and live cells at the optimal density are added to a well containing a final volume of 100 μL of T cell culture medium and then incubated at room temperature for 30 minutes.

[0451] The E-plate is then placed back on the analyzer and cell index values ​​are collected until a stable cell index is observed, at which point the program is paused and the E-plate is removed from the RTCA SP apparatus. The treatments are added to the appropriate wells. The treatments consist of 100 μL of T cell culture medium (untreated control) or the same volume containing effector cells or 0.5% Triton-x-100 (complete lysis control). Effector cell counts are performed using a quantitative method that can distinguish between dead and apoptotic cells. The values ​​for effector: target cells vary depending on the experiment.

[0452] During data analysis, the cell index was normalized using the following equation in the RTCA software package: NormalizedCIti=CIti / CInml_time

[0453] Where CIti = Cell Index (CI) at a specific time point, and CInml_time = CI at the time point before addition of T cells.

[0454] The data were then further processed relative to a fully lysed control to give % lysis: %Cytolysisst=[1-(NCIst) / (AvgNCIRt)]x100

[0455] Where NCIst is the normalized cell index of the sample, and NCIRt is the average of the normalized cell index of the matched reference wells.

[0456] 5 × 10 cells / mL were cultured in triplicate wells of a 96-well U-bottom plate in the absence of exogenous IL-2. 4 Repetitive stimulation assays were performed by mixing CoStAR-transduced or mock-transduced cells with LoVo-OKT3 cells at an E:T ratio of 8:1. T cells were assessed by flow cytometry using an αCD2-gated population, and fresh tumor cells were added at 7-day intervals, with T cell counts performed on days 1, 4, and 7. Relative expansion was assessed by well segmentation and counting based on the fold change relative to the original seeding density, taking into account the proportion of cells removed for counting.

[0457] result

[0458] A model system was developed t...

Claims

1. A protein comprising a consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 An amino acid sequence of a sequence of , wherein: a.X1 is any amino acid, b. X2 is any amino acid, c. X3 is P, X4 is any amino acid, d.X5 is Q, e.X6 is C or A, f.X7 is any amino acid, g.X8 is any amino acid, h.X9 is any amino acid, iX 10 is any amino acid, and JXj 11 is any amino acid.

2. The protein of claim 1, wherein the protein is part of a TRAF domain.

3. The protein of any one of the preceding claims, wherein the protein is part of a TRAF2 domain.

4. A protein according to any one of the preceding claims, wherein: a. The sequence comprises: i.X1 is S or P, ii. X2 is V, H or Y, iii.X4 is I, Q, V, iv.X7 is T or S, v.X8 is D, vi.X9 is K, D or G, vii.X 10 is T, S, G, or A, and viii.X 11 is D, S, N or D; or b. The sequence is at least 60% identical to a).

5. Proteins containing consensus B or X1X2X3X4X5X6X7X s X9X 10 X 11 An amino acid sequence of a sequence of , wherein: a.X1 is any amino acid, b. X2 is any amino acid, c.X3 is P, S, A or T, d.X4 is any amino acid, e.X5 is Q or E, f.X6 is E, g.X7 is any amino acid, h.X8 is any amino acid, i.X9 is any amino acid, JXj 10 is any amino acid, and k 11 is any amino acid.

6. The protein of claim 5, wherein the protein is part of a TRAF domain.

7. The protein of any one of the preceding claims, wherein the protein is part of a TRAF2 domain.

8. The protein of any one of the preceding claims, wherein: a. The sequence comprises: i.X1 is P, A, M, T, S, R, V, H, ii. X2 is F, A, L, I, T, V, G, C, A, F, P, iii.X4 is K, V, I, H, A, Q, T, E, S, iv.X7 is C, T, E, D, S, A, v.X8 is A, L, G, Y, I, Q, D, E, vi.X9 is F, H, K, R, P, A, G, Y, E, N, vii.X 10 are R, G, E, K, A, S, D, C, C, P, V, and viii.X 11 is S, C, D, P, W, T, A, G, E; or b. The sequence is at least 60% identical to a).

9. The protein of any one of the preceding claims, wherein the sequence is part of a Traf 2 / 3 sequence.

10. A protein comprising an amino acid sequence comprising a consensus C or X1X2X3X4X5X6 sequence, wherein: a.X1 is P, b. X2 is any amino acid, c.X3 is E, d.X4 is any amino acid, e. X5 is any amino acid, and f.X6 is Ac / Ar.

11. The protein of claim 5, wherein the protein is part of a TRAF domain.

12. The protein of any one of the preceding claims, wherein the protein is part of a TRAF6 domain.

13. A protein as claimed in any one of the preceding claims, wherein a. The sequence comprises: i.X2 is Q, P, E, V, T or S, ii.X4 is I, L, M, N, S, T, N, D, iii. X5 is N, D, R, S, G or Y; or b. The sequence is at least 60% identical to a).

14. An amino acid sequence comprising: d) one or more of SEQ ID NO: 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).

15. A TRAF domain in a CD40 protein, wherein the TRAF domain comprises one or more of the following: d) SEQ ID NO: 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).

16. A TRAF6 domain sequence comprising: PQEINF, PEEMSW, PPENYE or PQENSY.

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

18. A TRAF2 domain sequence comprising: SVQE, AVEE or PEEE.

19. A TRAF 6 or TRAF2 / 3 domain comprising: d) an amino acid sequence of one of SEQ ID NO: 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).

20. The protein, amino acid sequence or TRAF domain of any preceding claim, wherein the sequence is within the CD40 protein sequence.

21. The protein, amino acid sequence or TRAF domain of any one of the preceding claims, wherein the sequence is within a CD40 protein and is located at the corresponding TRAF2, TRAF2 / 3 and / or TRAF6 domain within the CD40 protein. 22.TRAF2 / TRAF3 domains containing consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of a.X1 is any amino acid, b. X2 is any amino acid, c. X3 is P, X4 is any amino acid, d.X5 is Q, e.X6 is C or A, f.X7 is any amino acid, g.X8 is any amino acid, h.X9 is any amino acid, iX 10 is any amino acid, and JXj 11 is any amino acid. 23.TRAF2 / TRAF3 domains containing consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of a.X1 is any amino acid, b. X2 is any amino acid, c.X3 is P, S, A or T, d.X4 is any amino acid, e.X5 is Q or E, f.X6 is E, g.X7 is any amino acid, h.X8 is any amino acid, i.X9 is any amino acid, JXj 10 is any amino acid, and k 11 is any amino acid.

24. TRAF6 domain comprising a consensus amino acid sequence of C or X1X2X3X4X5X6, wherein a.X1 is P, b. X2 is any amino acid, c.X3 is E, d.X4 is any amino acid, e. X5 is any amino acid, and f.X6 is Ac / Ar.

25. A CD40 domain comprising an amino acid sequence of one or more of the following: a. TRAF2 / TRAF3 domain, which contains consensus A or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of i.X1 is any amino acid, ii. X2 is any amino acid, iii. X3 is P, X4 is any amino acid, iv.X5 is Q, v.X6 is C or A, vi. X7 is any amino acid, vii. X8 is any amino acid, viii.X9 is any amino acid, ix.X 10 is any amino acid, and xX 11 is any amino acid. b. TRAF2 / TRAF3 domain, which contains consensus B or X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence of i.X1 is any amino acid, ii. X2 is any amino acid, iii. X3 is P, S, A or T, iv. X4 is any amino acid, v.X5 is Q or E, vi.X6 is E, vii. X7 is any amino acid, Viii.X8 is any amino acid, ix.X9 is any amino acid, xX 10 is any amino acid, and xi.X 11 is any amino acid. c. TRAF 6 domain comprising a consensus amino acid sequence of C or X1X2X3X4X5X6, wherein i.X1 is P, ii. X2 is any amino acid, iii.X3 is E, iv. X4 is any amino acid, v. X5 is any amino acid, and vi. X6 is Ac / Ar.

26. A chimeric co-stimulatory antigen receptor (CoStAR) comprising: an extracellular binding domain that binds to carcinoembryonic antigen (CEA) or an extracellular binding domain that binds to mesothelin (MSLN); a transmembrane domain connected 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 sequence of a variant TRAF2 / TRAF3 sequence or a variant TRAF6 sequence or a variant TRAF2 sequence, wherein the variant CD40 does not comprise SEQ ID NO:

42.

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

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

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

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

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

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

33. 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. The CoStAR of claim 29, wherein the extracellular binding domain comprises a 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 the CoStAR of any one of claims 1 to 34.

38. The cell of claim 37, wherein the cell comprises an α-β T cell, a γ-δ T cell, a regulatory T cell, a TIL, a NKT cell, or a NK cell.

39. The cell of claim 37, wherein the cell co-expresses CAR or TCR.

40. A method for preparing the cell of claims 37-39, comprising the step of transducing or transfecting the cell with the vector of claim 36.

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

42. A cell population enriched for cells expressing the CoStAR of any one of claims 26-34.

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

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

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

510.

46. ​​The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

511.

47. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

512.

48. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

513.

49. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

514.

50. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

515.

51. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

516.

52. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

517.

53. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO:

518.

54. The protein of claim 44, wherein the protein comprises the sequence of SEQ ID NO: 519.

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