Multispecific chimeric antigen receptor (CAR)-t cells directed to CD70 and methods of use thereof

Abstract

Aspects of the disclosure include dual chimeric antigen receptors (CARs) comprising a full-length CD27 receptor which binds CD70, polynucleotides encoding the same, multispecific CAR T cells comprising the polynucleotides and / or CARs, and methods of treating disorders (e.g., autoimmune diseases or cancer) in a patient using the same.

Description

MULTISPECIFIC CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELLS DIRECTED TO CD70 AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application Ser. No. 63 / 529,673 filed on July 28, 2023, the disclosure of which is expressly incorporated by reference herein in its entirety. FIELD OF DISCLOSURE

[0002] The present disclosure relates generally to chimeric antigen receptors (CARs) comprising an antigen binding molecule which binds CD70, polynucleotides encoding the same, multispecific CAR T cells comprising the polynucleotides and / or CARs, and methods of treating disorders associated with CD70 expression in a patient using the same. BACKGROUND OF THE DISCLOSURE

[0003] T-cell-engaging therapies employing chimeric antigen receptor (CAR) T cells have opened up new frontiers in cancer immunotherapy (June et al., Science 2018; 359: 1361–1365) and, more recently, in treating autoimmune diseases (Bao L. et al., Zool Res.2022 Mar 18; 43(2): 150–165). Unfortunately, however, the duration of clinical responses obtained with these types of therapies has thus far been limited, despite initial high remission rates. In adult B-ALL, a median event-free survival of 3–6 months has been reported, while more than half of the patients with aggressive non-Hodgkin’s lymphoma (NHL) relapse within 1 year after therapy (Shah et al. J. Clin. Oncol.202139:1650–59; Chong et al. N. Engl. J. Med.2021384:673–74). Even in patients with initial objective responses, the long-term success rate remains unknown, with only limited follow-up to date. Accordingly, and despite rapid and intense development of CAR T therapies, the optimal parameters for CAR T cell-target interactions that will result in sustained in vivo and in-human efficacy are not yet well understood.

[0004] One attractive target is human cluster of differentiation 70 (CD70). CD70 is a transmembrane glycoprotein and a member of the tumor necrosis factor (TNF) superfamily, and is the only known ligand for CD27. CD70 displays aberrant constitutive expression in a variety of cancers, including renal cell carcinoma, leukemia, non-small cell lung cancer, melanoma,glioblastoma, and others. In addition, the CD27-CD70 pathway has been suggested to play a role in autoimmune diseases (Han BK et al., Semin Arthritis Rheum.2016 Feb;45(4):496-501).

[0005] The first-generation CD70-targeted CAR utilized a full-length CD27 protein targeting B cell malignancies (Shafer et al., Blood 2011; 117: 4304-4314). Subsequently, improved efficacy was demonstrated with a truncated CD27 protein (Wang et al., Clin. Cancer Res.2017; 23: 2267- 2276). This CAR was brought to a phase I clinical trial for solid tumors (NCT02830724) and has been demonstrated to be effective against head and neck cancers and gliomas expressing CD70. To date, CD70-targeted CARs have been examined in the context of alpha beta (αβ) T cells derived from peripheral blood T cells. Unfortunately, however, the effective use of αβ T cells is complicated by their high alloreactive potential in general, and by their propensity to trigger cytokine release syndrome (CRS) in particular.

[0006] Gamma delta (γδ) T cells are thymus-derived lymphocytes that differ from αβ T cells in their mechanism of activation and function, as well as in their anatomical distribution. In particular, while αβ T cells function exclusively in adaptive immunity, γδ T cells are innate-like immune cells that recognize disease-associated cells through a repertoire of activating receptors in a MHC-independent manner, similar to Natural Killer cells (NK cells) (Welsh et al., Immunol Rev. 1997; 159: 79-93). As such, and in contrast to αβ T cells, γδ T cells can potentially be used in an allogeneic setting without the risk of causing graft versus host disease (GvHD). Moreover, recent studies have suggested that engineered γδ T cells may produce less proinflammatory cytokines than αβ-T cells, which could thereby reduce the risk of CRS in patients (Harrer et al., BMC Cancer 2017; 17(1): 551). Given the differences in mechanism of action and function for αβ T cells as compared to γδ T cells, however, the practical translation of the current CD70-targeted CAR T therapeutic approaches to γδ T cells is at best uncertain.

[0007] Notably, persistence and toxicity both remain as major issues for CAR T therapies targeting CD70, and prior art attempts to address these issues have met with mixed success. For example, TALEN® gene editing was employed in the anti-CD70 allogeneic CD70-targeted CAR T-cell treatment ALLO-316 to disrupt TCR alpha and knock out CD52, to allow for co- administration of an anti-CD52 antibody to deplete host T-cells and promote the persistence of the allogeneic CAR T-cells, and to reduce the risk of graft-versus-host disease (GVHD). Unfortunately, however, 65% of patients still experienced cytokine release syndrome with thecombination therapy, and the maximum tolerated dose remains under investigation. (Meng et al., Cells 2024 Jan; 13(1): 34.

[0008] What is needed, then, are new approaches to improve T cell activation and persistence, overcome tumor immunosuppression, address tumor escape, mitigate toxicities, and increase patient access. The present invention addresses these and other unmet needs. SUMMARY OF DISCLOSURE

[0009] The present disclosure provides dual chimeric antigen receptors (CARs) binding to CD70 and additional disease-associated antigens of interest, including tumor-associated antigens, autoantigens, and pathogenic antigens, multispecific CAR T cells comprising same, and uses thereof. As demonstrated herein, incorporating a CD70 binding domain into the subject multispecific CAR T cells can target CD70+ activated alloreactive host T cells and limit the host vs. graft (HvG) Vδ1 T cell rejection to aid in CAR persistence. Moreover, as further demonstrated herein, the use of full-length CD27 receptor for CD70 targeting improves both the viability and tumoricidal activity of the resulting multispecific CAR T cells. In embodiments, the multispecific CAR T cell is a γδ T cell, preferably wherein the γδ T cell is a δ1 γδ T cell.

[0010] In one aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a dual chimeric antigen receptor (CAR) comprising: a first chimeric antigen receptor (CAR), wherein the first CAR comprises a full length CD27 receptor, a first costimulatory domain, and a first intracellular signaling domain; a cleavage sequence; and a second CAR, wherein the second CAR comprises an affinity binding domain that binds to at least one disease-associated antigen of interest, a second costimulatory domain, and a second intracellular signaling domain.

[0011] In embodiments, the isolated nucleic acid encodes a polypeptide comprising, from C- terminus to N-terminus, the first CAR, the cleavage sequence, and the second CAR. In embodiments, the isolated nucleic acid encodes a polypeptide comprising, from C-terminus to N- terminus, the second CAR, the cleavage sequence, and the first CAR.

[0012] In embodiments, the cleavage sequence is a 2A self-cleaving sequence selected from T2A, P2A, E2A, and F2A self-cleaving sequences. In embodiments, the 2A self-cleaving sequence is a P2A self-cleaving sequence; optionally wherein the P2A self-cleaving sequence encoded by the isolated nucleic acid has an amino acid sequence as set forth in SEQ ID NO: 8, 68, or 69.

[0013] In embodiments, the second CAR further comprises a hinge domain; optionally wherein the hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or a receptor-derived hinge. In embodiments, the receptor-derived hinge is a CD8 alpha hinge domain, optionally wherein the CD8 alpha hinge domain comprises an amino acid sequence set forth as SEQ ID NO: 97.

[0014] In embodiments, the second CAR further comprises a transmembrane (TM) domain; optionally wherein the TM domain comprises a TM region of 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM- 1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB- A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. In embodiments, the TM domain comprises a TM domain of CD8, preferably wherein the CD8 TM domain is a TM domain of CD8 alpha; optionally wherein the TM domain comprises the amino acid sequence set forth as SEQ ID NO: 99.

[0015] In embodiments, the first and / or second costimulatory domain comprises a costimulatory domain of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80,CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (OX40), CD137 (4- 1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, or TRANCE / RANKL, or a portion thereof, or combinations thereof. In embodiments, the first and / or second costimulatory domain is 4-1BB; optionally wherein the 4-1BB costimulatory domain comprises an amino acid sequence set forth as SEQ ID NO: 4.

[0016] In embodiments, the first and / or second intracellular signaling domain is selected from CD3ζ, DAP12, LFA-1, and repeat (2-5) DAP10 YINM motifs. In embodiments, the first and / or second intracellular signaling domain is CD3ζ.

[0017] In embodiments, the second CAR is a tandem CAR.

[0018] In embodiments, the at least one disease-associated antigen is a tumor antigen, an autoantigen, or a pathogenic antigen. In embodiments, the disease-associated antigen is selected from the group comprising or consisting of CD19, CD20, CD30, CD22, CD33, CD37, CD38, CD56, CD79b, CD123, CD138, CD123, CD79b, CD75, CA6, GPRC50, TAC1, GD2, B7H6, B- cell maturation antigen (BCMA), B-cell activating factor receptor (BAFF-R), Fc Receptor-Like 5 (FcRL5), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-1, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c- Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, L1CAM, GPC2, GPC3, folate binding protein / receptor, SLC44A4, Cripto, CTAGIB, AXL, IL-13R, IL-3Rα2, SLTRK6, gp100, MART1, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3. MAGEA4), KKLC1, mutated ras, BRAF, p53, MHC class I chain-related molecule A (MICA), or MHC class I chain-related molecule B (MICB), and one or more antigens of HPV, CMV, or EBV.

[0019] In embodiments, the disease-associated antigen is selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123,CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In embodiments, the disease-associated antigen is a B cell antigen selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In embodiments, the disease-associated antigen is a plasma cell antigen selected from the group comprising or consisting of CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB.

[0020] In embodiments, the isolated nucleic acid further encodes one or more additional polypeptides. For example, the isolated nucleic acid further encodes dominant-negative TGFβ receptor II (dnTGFβR2), e.g., the amino acid sequence as set forth in SEQ ID NO: 22.

[0021] In embodiments, the second CAR comprises an affinity binding domain that binds to CD19. In embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 117. In embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 121.

[0022] In another aspect, the present disclosure provides a recombinant expression vector comprising the nucleic acid herein.

[0023] In another aspect, the present disclosure provides an isolated host cell comprising the recombinant expression vector herein.

[0024] In another aspect, the present disclosure provides a multispecific CAR T cell having improved and / or increased persistence, viability and / or tumoricidal activity comprising: a first chimeric antigen receptor (CAR), wherein the first CAR comprises full length CD27 receptor (SEQ ID NO: 32), a first costimulatory domain, and a first intracellular signaling domain; and a second CAR, wherein the second CAR comprises an affinity binding domain that binds to at least one disease-associated antigen of interest, a second costimulatory domain, and a second intracellular signaling domain, wherein the multispecific CAR T cell functionally expresses the CD27 receptor and the affinity binding domain on surface of the T cell. In embodiments, the multispecific CAR T cell is a γδ T cell, preferably wherein the γδ T cell is a δ1 γδ T cell.

[0025] In embodiments, the second CAR further comprises a hinge domain; optionally wherein the hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or a receptor-derived hinge.In embodiments, the receptor-derived hinge is a CD8 alpha hinge domain, optionally wherein the CD8 alpha hinge domain comprises an amino acid sequence set forth as SEQ ID NO: 97.

[0026] In embodiments, the second CAR further comprises a transmembrane (TM) domain; optionally wherein the TM domain comprises a TM region of 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM- 1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB- A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. In embodiments, the TM domain comprises a TM domain of CD8, preferably wherein the CD8 TM domain is a TM domain of CD8 alpha; optionally wherein the TM domain comprises the amino acid sequence set forth as SEQ ID NO: 99.

[0027] In embodiments, the first and / or second costimulatory domain comprises a costimulatory domain of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (OX40), CD137 (4- 1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI,FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, or TRANCE / RANKL, or a portion thereof, or combinations thereof. In embodiments, the first and / or second costimulatory domain is 4-1BB; optionally wherein the 4-1BB costimulatory domain comprises an amino acid sequence set forth as SEQ ID NO: 4.

[0028] In embodiments, the first and / or second intracellular signaling domain is selected from CD3ζ, DAP12, LFA-1, and repeat (2-5) DAP10 YINM motifs. In embodiments, the first and / or second intracellular signaling domain is CD3ζ.

[0029] In embodiments, the second CAR is a tandem CAR.

[0030] In embodiments, the at least one disease-associated antigen is a tumor antigen, an autoantigen, or a pathogenic antigen. In embodiments, the disease-associated antigen is selected from the group comprising or consisting of CD19, CD20, CD30, CD22, CD33, CD37, CD38, CD56, CD79b, CD123, CD138, CD123, CD79b, CD75, CA6, GPRC50, TAC1, GD2, B7H6, B- cell maturation antigen (BCMA), B-cell activating factor receptor (BAFF-R), Fc Receptor-Like 5 (FcRL5), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-1, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c- Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, L1CAM, GPC2, GPC3, folate binding protein / receptor, SLC44A4, Cripto, CTAGIB, AXL, IL-13R, IL-3Rα2, SLTRK6, gp100, MART1, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3. MAGEA4), KKLC1, mutated ras, BRAF, p53, MHC class I chain-related molecule A (MICA), or MHC class I chain-related molecule B (MICB), and one or more antigens of HPV, CMV, or EBV.

[0031] In embodiments, the disease-associated antigen is selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In embodiments, the disease-associated antigen is a B cell antigen selected from the group comprising or consisting ofCD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In embodiments, the disease-associated antigen is a plasma cell antigen selected from the group comprising or consisting of CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB.

[0032] In embodiments, the isolated nucleic acid further encodes one or more additional polypeptides. For example, the isolated nucleic acid further encodes dominant-negative TGFβ receptor II (dnTGFβR2), e.g., the amino acid sequence as set forth in SEQ ID NO: 22.

[0033] In embodiments, the second CAR comprises an affinity binding domain that binds to CD19. In embodiments, the first CAR comprises the amino acid sequence set forth in SEQ ID NO: 138 and the second CAR comprises the amino acid sequence set forth in SEQ ID NO: 139.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of multispecific CAR T cells herein, the plurality comprising at least about 107T cells, preferably from about 108T cells to about 1011T cells; optionally further comprising a pharmaceutically acceptable excipient. In embodiments, the plurality comprises a composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2- γδ T cells, most preferably δ1 γδ T cells.

[0035] In another aspect, the present disclosure provides a method of treating an autoimmune disease in a subject in need thereof, comprising: administering a therapeutically effective amount of multispecific CAR T cells, or a pharmaceutical composition herein. In embodiments, the method comprises performing a plurality of administrations of the multispecific CAR T cells, wherein the interval between the plurality of administrations is at least about a week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or wherein the plurality of administrations is no more than once every 6 or 12 months. In embodiments, the autoimmune disease is lupus, lupus nephritis, systemic sclerosis, idiopathic inflammatory myopathies, or myasthenia gravis. In embodiments, the autoimmune disease is lupus.

[0036] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising: administering a therapeutically effective amount of multispecific CAR T cells or a pharmaceutical composition herein , wherein the cancer comprises hematological and / or solid tumor cells that exhibit cell surface expression of CD70. In embodiments, the method comprises performing a plurality of administrations of the multispecific CAR T cells, wherein the interval between the plurality of administrations is at least about a week,preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or wherein the plurality of administrations is no more than once every 6 or 12 months.

[0037] In another aspect, the present disclosure provides use of the multispecific CAR T cell, or the pharmaceutical composition herein, in the manufacture of a medicament for the treatment or prevention of autoimmune disease or cancer. INCORPORATION BY REFERENCE

[0038] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG.1 is a graph illustrating CD70 expression in different cancer cell lines.

[0040] FIGS. 2A-2C depict graphs illustrating in vitro cytotoxicity of Vδ1 CD27 receptor CAR T cells against various cancer cell lines.

[0041] FIG.3 is a graph illustrating that in vitro cytotoxicity of Vδ1 CD27 receptor CAR T cells against A498 cells is not affected by the presence of soluble CD27.

[0042] FIG. 4 depicts graphs illustrating in vivo therapeutic efficacy of Vδ1 CD27 receptor CAR T cells in a subcutaneous human xenograft A498 cell NOD scid gamma (NSG) mouse model.

[0043] FIG. 5 depicts graphs illustrating in vivo therapeutic efficacy of Vδ1 CD27 receptor CAR T cells in a subcutaneous human xenograft ACHN cell NSG mouse model.

[0044] FIG.6 depicts a series of plots illustrating the transduction efficiency of Vδ1 T cells by staining for P2A as a method for CAR detection.

[0045] FIG.7 is a graph illustrating the gene knockout efficiency of two different guide RNAs targeting cytokine inducible SH2 containing protein (CISH) in Vδ1 T cells.

[0046] FIG.8A is a graph illustrating Vδ1 T cell enrichment post-CISH gene knockout from two different guide RNAs.

[0047] FIG.8B is a graph illustrating cell viability of Vδ1 T cells post CISH gene knockout from two different guide RNAs.

[0048] FIG. 9 illustrates the transduction efficiency of ADI-270 by staining for P2A as a method for CAR detection.

[0049] FIG.10 illustrates CD70 expression in different cancer cell lines.

[0050] FIG.11 illustrates in vitro cytotoxicity of ADI-270 against various cancer cell lines.

[0051] FIG.12 illustrates in vitro cytotoxicity of ADI-270 in the presence of TGFβ1.

[0052] FIG.13 illustrates in vitro proliferation of ADI-270 in the presence of TGFβ1.

[0053] FIG.14 illustrates intracellular SMAD2 / 3 phosphorylation of ADI-270 in the presence of TGFβ1.

[0054] FIG. 15 illustrates ADI-270 was armored against TGF-β1-mediated transcript alterations.

[0055] FIGS.16A-16B illustrate ADI-270 cytokine profile with proinflammatory and chemo- attractive cytokines.

[0056] FIGS. 17A-17B illustrate in vivo therapeutic efficacy of ADI-270 in a subcutaneous human xenograft A498 cell NSG mouse model.

[0057] FIGS. 18A-18B illustrate in vivo therapeutic efficacy of ADI-270 in a subcutaneous human xenograft A498 cell NSG mouse model.

[0058] FIGS. 19A-19B illustrate in vivo therapeutic efficacy of ADI-270 in a subcutaneous human xenograft HuT78 cell NSG mouse model.

[0059] FIGS. 20A-20C illustrate ADI-270 homed to the tumor, proliferates, and expresses activation markers from ex vivo tissue analysis.

[0060] FIGS.21A-21B illustrate expansion profile of Vδ1 T cells engineered to express the CD27 receptor CAR compared to anti-CD70 scFv based CARs.

[0061] FIGS.22A-22B illustrate in vitro cytotoxicity of ADI-270 compared to αβ CAR T cells engineered to express anti-CD70 scFv CARs.

[0062] FIGS. 23A-23C illustrate in vivo therapeutic efficacy of ADI-270 in subcutaneous human hematologic-derived tumor xenograft NSG mouse models.

[0063] FIG. 24 illustrates Host vs Graft (HvG) armoring supported improved persistence of ADI-270.

[0064] FIG.25 Illustrates HvG armoring of ADI-270 against primed alloreactive T cells.

[0065] FIG.26A shows the design of CAR construct PL1605. FIG.26B shows the design of CAR constructs PL1626 and PL1627.

[0066] FIG.27A shows the yield of δ1 γδ T cells expressing exemplary CARs compared head- to-head with ADI-001. FIG.27B shows the growth of δ1 γδ T cells expressing exemplary CARs in extended cultures.

[0067] FIGS.28A-28B shows the expression of exemplary CARs in transduced δ1 γδ T cells.

[0068] FIG. 29 shows cytotoxicity of δ1 γδ T cells expressing exemplary CARs tested with tumor cell lines. The data shown is averaged across 2 donors. The E:T ratios are 2:1 for all cell lines except A498, for which the E:T ratio is 1:1.

[0069] FIGS. 30A and 30B show an exemplary CARs was functionally equivalent to anti- CD19 CAR against healthy B cells and additionally targeted activated primary T cells. For FIG. 30A, effectors from 2 donors were cultured for 24 hours with PBMCs at E:T of 1:1. For FIG.30B, effectors from 5 donors were co-cultured for 24 hours with donor mismatched PBMCs stimulated with CD3 / CD28 at E:T of 2:1.

[0070] FIG. 31 shows that an exemplary CAR was equivalent to anti-CD19 CAR in cytotoxicity against B cells from patient with multiple sclerosis. Effectors from 2 donors co- cultured for 24 hours with patient PBMCs at E:T of 1:2 (T = CD19+).

[0071] FIG.32 illustrates the CAR-mbIL-12 constructs designs.

[0072] FIGS.33A-33D illustrate the expansion and expression of the CAR-mbIL-12 in Vδ1 T cells.

[0073] FIG.34 illustrates the enhanced in vitro cytotoxicity of CAR-mbIL-12 in Vδ1 T cells.

[0074] FIG 35. illustrates in vivo therapeutic efficacy of CAR-mbIL-12 in Vδ1 T cells in a subcutaneous human xenograft Raji cell NSG mouse model.

[0075] FIGS.36A-36B. illustrate CISH KO enhanced the in vitro cytotoxicity of Vδ1 T cells.

[0076] FIGS. 37A-37B. illustrate CBL-B KO enhanced the in vitro cytotoxicity of Vδ1 T cells.

[0077] FIGS. 38A-38B. illustrate Roquin KO enhanced the in vitro cytotoxicity of Vδ1 T cells.

[0078] FIGS.39A-39B. illustrates CD58 or ICAM-1 KO enhanced the in vitro cell survival of Vδ1 T cells in an allogeneic MLR assay.DETAILED DESCRIPTION I. DEFINITIONS

[0079] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0080] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0081] As used herein, “w / v” refers to the weight of the component in a given volume of solution.

[0082] “Ranges”: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0083] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal amenable to the methods described herein. In certain non- limiting embodiments, the patient, subject or individual is a human.

[0084] The term “diagnosis”, or “diagnosing” as used herein refers to the process of identifying a disease, such as autoimmune disease or cancer, by its signs, symptoms, and / or results of various tests. A conclusion reached through such a process is a diagnosis. Forms of testing commonly performed include blood tests, medical imaging, urinalysis, biopsy, and the like.

[0085] As used herein, the term “agent” refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell (e.g., cell engineered to express a chimeric antigen receptor). Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional agent induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject suffering from autoimmune disease or cancer, or other disease / condition.

[0086] The term "therapeutically effective amount", or simply “effective amount” refers to the amount of an agent or composition (e.g., composition comprising an agent) that will elicit a biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of an agent, or a composition comprising an agent, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease (e.g., autoimmune disease or tumor) being treated. The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0087] The term “γδ T cells (gamma delta T cells)” as used herein refers to a subset of T cells that express a distinct T-cell receptor (TCR), namely γδ TCR, on their surface, composed of one γ-chain and one δ-chain. The term “γδ T cells” specifically includes all subsets of γδ T cells, including, without limitation, Vδ1 and Vδ2, Vδ3 γδ T cells, as well as naïve, effector memory, central memory, and terminally differentiated γδ T cells. As a further example, the term “γδ T cells” includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells. In embodiments, the γδ T-cells are Vδ1-, Vδ2-, or Vδ1- and Vδ2-. Compositions and methods for making and using engineered and non-engineered γδ T cells and / or sub-types thereof include, without limitation, those described in US 2016 / 0175358; WO 2017 / 197347; US 9499788; US 2018 / 0169147; US 9907820; US 2018 / 0125889 and US 2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including the said compositions and methods for making and using engineered and non-engineered γδ T cells and / or sub-types thereof. The present application further contemplates T cells, or other engineered leukocytes or lymphocytes, that express one γ-chain or one δ-chain, optionally incombination with a second polypeptide to form a functional TCR. Such engineered leukocytes or lymphocytes, that express one γ-chain or one δ-chain may be used in the methods or present in the compositions described herein.

[0088] The term “αβ T cell” refers to T cells expressing α and β chains of the TCR as part of a complex with CD3 chain molecules. Each α and β chain contains one variable and one constant domain. αβ T cells primarily recognize peptide antigens presented by major histocompatibility complex (MHC) class I and class II molecules, where most of the receptor diversity is contained within the third complementarity determining region (CDR3) of the TCR α and β chains.

[0089] As used herein, the term “T lymphocyte” or “T cell” refers to an immune cell that expresses or has expressed CD3 (CD3+) and a T Cell Receptor (TCR+). T cells play a central role in cell-mediated immunity. A T cell that “has expressed” CD3 and a TCR has been engineered to eliminate CD3 and / or TCR cell surface expression.

[0090] A “multispecific CAR T cell” refers to a T cell expressing two or more CARs. In the context of a multispecific CAR T cell, as used herein, the term “the first CAR” refers to a CAR comprising full-length CD27 for targeting CD70, and the term “the second CAR” refers to a CAR comprising one or more affinity binding domains for targeting one or more disease-associated antigens other than CD70.

[0091] As used herein, the term “TCR” or “T cell receptor” refers to a dimeric heterologous cell surface signaling protein forming an alpha-beta or gamma-delta receptor or combinations thereof. αβTCRs recognize an antigen presented by an MHC molecule, whereas γδ TCR can recognize an antigen independently of MHC presentation.

[0092] As used herein, the term “CD70” refers to a molecule that belongs to the tumor necrosis factor (TNF) ligand family, and is also known as CD27LG and TNFSF7. CD70 is a ligand for CD27 (TNFRSF27). The term CD70 can include, but is not limited to, native CD70, an isoform of CD70, or an interspecies CD70 homolog of CD70. The amino acid sequence of human CD70 (hCD70) is provided in NCBI Accession NP 001243.1 (GI: 4507605) (SEQ ID NO: 1). Residues 1-17 of SEQ ID NO: 1 correspond to the cytoplasmic region of hCD70, residues 18-38 correspond to the transmembrane region of hCD70, and residues 39-193 correspond to the extracellular region of hCD70. At least one other isoform of hCD70 is known and is identified by the UniProt identifier P32970-2. As used herein, CD70 includes human CD70 and non-human CD70 homologs, as well as variants, fragments, or post-translationally modified forms thereof, including, but not limitedto, N- and O-linked glycosylated forms of CD70. CD70 proteins can further include fragments comprising all or a portion of SEQ ID NO: 1 (e.g., amino acids 39-193 of SEQ ID NO: 1, corresponding to an extracellular component of hCD70, amino acids 18-193, corresponding to a transmembrane and extracellular component of hCD70).

[0093] As used herein, the term “CD19” refers to a type-I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD19 is expressed on B cells throughout most stages of B-cell differentiation, including, pre-B cells, B cells in early development (e.g., immature B cells), mature B cells through terminal differentiation into plasma cells, and malignant B cells. The term “CD19” can include, but is not limited to, native CD19, an isoform of CD19, or an interspecies CD19 homolog of CD19. The amino acid sequence of human CD19 (hCD19) is provided in SwissPort Accession No. P15391 (SEQ ID NO: 96). As used herein, CD19 includes human CD19 and non-human CD19 homologs, as well as variants, fragments, or post- translationally modified forms thereof, including, but not limited to, N- and O-linked glycosylated forms of CD19. CD19 proteins can further include fragments comprising all or a portion of SEQ ID NO: 96.

[0094] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encodes cell-surface antigen-presenting proteins. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. Herein, the abbreviations MHC or HLA are used interchangeably.

[0095] “Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0096] The terms “binding domain, “affinity binding domain” or “means for specifically binding” as used herein refer to a binding moiety which binds to a specific antigen with a higher affinity than to a non-specific antigen and is endowed with an affinity of at least 10-6M, as determined by assays which are well known in the art, including surface plasmon resonance (SPR). According to a specific embodiment, the affinity is 500 nM-0.01 nM, 100 nM-0.01 nM, 50 nM- 0.01 nM, 10 nM-0.01 nM, 5 nM-0.01 nM. In embodiments, the affinity binding domain or means for specifically binding recognizes a disease-associated antigen, e.g., a tumor antigen, an autoimmune antigen, or a pathogenic antigen.

[0097] In embodiments, the affinity binding domain or means for specifically binding is an antibody. The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, intact antibodies, antibody fragments, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab), as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0098] An “intact” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[0099] “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or one or more variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10): 1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In embodiments, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. Also included among antibody fragments are portions of antibodies (and combinations of portions of antibodies, for example, scFv) that may be used as targeting arms, directed to an epitope, in chimeric antigenic receptors of the present disclosure. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target.

[0100] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab’ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0101] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0102] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0103] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form a desired structure for antigen binding. For a review of sFv, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In one embodiment, an antibody derived scFv is used as the targeting arm of a modified T cell as disclosed herein. In terms of scFv antibody fragments, where a certain order of VH and VL region in the binding domain is explicitly or implicitly described, the present disclosure also includes thealternate embodiment in which the order of VH and VL regions are reversed, e.g., in an scFv or CAR comprising an scFv binding domain. Thus, description of a VH-VL order also describes the alternate VL-VH order, e.g., in an scFv or CAR comprising an scFv binding domain. Moreover, description of a VL-VH order also describes the alternate VH-VL order, e.g., in an scFv or a CAR comprising an scFv binding domain. VH and VL regions are either joined directly or joined by a peptide-encoding linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.

[0104] The scFv linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al, Anal. Chem.80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 124), (GGGS)n (SEQ ID NO: 125), and (GGGGS)n (SEQ ID NO: 126), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 127), GGSGG (SEQ ID NO: 128), GSGSG (SEQ ID NO: 129), GSGGG (SEQ ID NO: 130), GGGSG (SEQ ID NO: 131), GSSSG (SEQ ID NO: 132), GGGGS (SEQ ID NO: 133), GGGGSGGGGSGGGGS (SEQ ID NO: 123) and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention.

[0105] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including proteins or peptides, can serve as an antigen.

[0106] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant capable of specific binding to an immunoglobulin or receptor, e.g., CD27 binding domain. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0107] The term “specifically binds”, as used herein refers to a affinity binding entity which recognizes a specific molecule / ligand, but does not substantially recognize or bind othermolecules in a sample. For example, an affinity binding entity that specifically binds to a molecule from one species may also bind to that molecule from one or more other species. But, such cross-species reactivity does not itself alter the classification as specific. In another example, an affinity binding entity that specifically binds to a molecule may also bind to different allelic forms of the molecule. However, such cross reactivity does not itself alter the classification as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of a protein (or a peptide) with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an affinity binding entity recognizes and binds to a specific a structure rather than to proteins generally. If an affinity binding entity is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the affinity binding entity, will reduce the amount of labeled A bound to the affinity binding entity.

[0108] In embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10-8M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0109] The term “chimeric antigen receptors (CARs)” refers to artificial T cell receptors, T- bodies, single-chain immunoreceptors, chimeric T cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a one or more disease-associated antigens (e.g., an autoimmune disease-associated antigen or tumor associated antigen).

[0110] The term “dual CAR” refers to two CARs co-expressed in one cell that target different antigens. The two CARs may be encoded by multiple nucleic acid molecules. Alternately, the two CARs may be encoded by one nucleic acid molecule. A “dual CAR construct” is a nucleic acid molecule encoding two CARs.

[0111] The term “tandem CAR” refers to a CAR with multiple affinity binding domains. The multiple affinity binding domains may be connected by one or more linkers. Examples of dualCARs and tandem CARs designs include those described in Guedan S et al., Engineering and Design of Chimeric Antigen Receptors, Mol Ther Methods Clin Dev.2019 Mar 15; 12: 145–156, which is incorporated by reference herein in its entirety.

[0112] In embodiments, the CARs comprise an intracellular activation domain (allowing the T cell to activate upon engagement of targeting moiety with target cell, such as an autoimmune disease-associated cell or tumor cell), a transmembrane domain, and an extracellular domain that may vary in length and comprises a disease- or disorder-associated antigen. In embodiments, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise costimulatory domains for additional co-stimulatory signaling. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro- drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0113] The “costimulatory domain” in the context of a chimeric receptor, also referred to herein as a chimeric antigen receptor (CAR), of the present disclosure enhances cell proliferation, cell survival and development of memory cells for cytotoxic cells that express the chimeric receptor. The chimeric receptors of the invention may include one or more costimulatory domains selected from the costimulatory domains of proteins in the TNFR superfamily, CD28, CD137 (4- 1BB), CD134 (OX40), Dapl0, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD1 la / CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3, or combinations thereof. If the chimeric receptor includes more than one costimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. The costimulatory domain is an intracellular domain that may locate between an affinity binding domain and an intracellular signaling domain in the chimeric receptor.

[0114] The term “costimulatory domain” as used herein also encompasses any modifications thereof, examples of which are described in US Patent Application No.20200129554; US Patent Application No.20200317777; WO2019010383; Li, W., et al., (2020) Immunity 53: 456-470; andLi, G., et al., (2017) J Immunol 198(1 Supplement): 198.4, the contents of each of which are incorporated herein in their entirety.

[0115] The “intracellular signaling domain” in the context of a chimeric receptor of the present disclosure transduces the effector function signal and directs the cytotoxic cell to perform its specialized function, i.e., harming and / or destroying the target cells. Examples of suitable intracellular signaling domains include, e.g., the ζ chain of the T cell receptor complex or any of its homologs, e.g., η chain, FcsRly and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 ζ chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In embodiments, the intracellular signaling domain of a chimeric receptor may be human CD3 ζ chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors and combinations thereof.

[0116] The intracellular signaling domains may include intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (Park et al., "Are all chimeric antigen receptors created equal?" J Clin Oncol., vol. 33, pp. 651-653, 2015). Additional intracellular signaling domains include signaling domains used by NK and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immunol., vol. 6, p. 195, 2015) such as signaling domains of NKp30 (B7-H6) (Zhang et al., "An NKp30- based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo," J Immunol., vol.189, pp.2290-2299, 2012), and DAP12 (Topfer et al., "DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol., vol. 194, pp. 3201- 3212, 2015), NKG2D, NKp44, NKp46, DAP10, and CD3z. Additionally intracellular signaling domains also includes signaling domains of human Immunoglobulin receptors that contain immunoreceptor tyrosine based activation motif (ITAM) such as FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (Gillis et al., "Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies," Front Immunol., vol.5, p.254, 2014).

[0117] In embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, or CD66d. In exemplary embodiments the intracellular signaling domain in the chimeric receptor includes a cytoplasmic signaling domain of human CD3 ζ. The term “intracellular signaling domain” as used herein also encompasses any modifications examples of which are described in US PatentApplication No. 2020 / 0317777, as well as Navarro, P., and Reyburn, HT., (2009) J Biol Chem 284(24): 16463-16472; Giurisato, E., et al., (2007) Mol Cell Biol 27(24): 8583-8599; and Wu, J., et al., (2000) J Exp Med 192(7): 1059-1068, the contents of each of which are incorporated herein in their entirety.

[0118] The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0119] As used herein, the term “autologous” is meant to refer to any material derived from an individual which is later to be re-introduced into the same individual.

[0120] As used herein, the term “allogeneic” refers to material derived from an animal which is later introduced into a different animal of the same species.

[0121] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or condition by decreasing one or more signs or symptoms associated with the disease or condition, for example as compared to the response in the absence of the therapy. For example, administration of a therapeutic agent may provide an effect that decreases one or more signs or symptoms associated with an autoimmune disease or cancer.

[0122] As used herein, the term “administration” means to provide or give a subject one or more agents, such as an agent that treats one or more signs or symptoms associated with a condition / disorder or disease including but not limited to autoimmune disease, cancer (e.g., lymphoma), viral infection, bacterial infection, etc., by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0123] The term “pharmaceutically acceptable”, as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulatory agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate. For example, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an, e.g., a multispecific CAR T cell, preferably a multispecific γδ CAR T cell, as described herein.

[0124] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, usedas the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0125] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0126] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0127] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0128] “Expression cassette” refers to a nucleic acid comprising expression control sequences operatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression cassettes can be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be in a host cell, such as a T cell.

[0129] The term “autoimmune disease” refers to a disorder that results from an autoimmune response. An autoimmune disease may be the result of an inappropriately excessive response to an antigen associated with an autoimmune disease (e.g., a self-antigen).

[0130] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule which contains epitopes that stimulates a host's immune system to make a cellular antigen-specific immune response and / or a humoral antibody response against the disease. A disease-associated antigen may be an autoimmune disease-associated antigen, a tumor associated antigen, or a pathogenic antigen.

[0131] The term “autoimmune disease-associated antigen” or “autoantigen” encompasses endogenous antigens that stimulate the production of an autoimmune response, such as production of autoantibodies. Antigens associated with autoimmune diseases also include antigens from a normal tissue that is the target of a cell mediated or an antibody-mediated immune response that may result in the development of an autoimmune disease. The term “autoimmune disease- associated cell” refers to a cell involved in an autoimmune disease, e.g., the onset and / or development of an autoimmune disease. In embodiments, an autoimmune disease-associated cell is a B cell, a plasma cell, or a T cell. Examples of autoimmune disease-associated cells include pro B cells, pre B cells, transitional / immature B cells, naive B cells, GC B cells, memory B cells, plasmablasts, and plasma cells. Examples of autoimmune disease-associated cells also include T cells such as Th1 / Th17 T cells, CD4+ T cells, and keratinocytes.

[0132] The term “autoantibody” refers to an antibody produced by a host (with or without immunization) and directed to a host antigen.

[0133] The term “tumor-associated antigen” or “TAA” or “tumor antigen” is used herein to refer to a molecule or complex which is expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. Tumor-associated antigens may be antigens not normally expressed by the host; they may be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they may be identical to molecules normally expressed but expressed at abnormally high levels; or they may be expressed in a context or milieu that is abnormal. Tumor-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules. In embodiments, the tumor-associated antigens are expressed at least from about 5 to about 100-folds higher in tumor cells relative to the levels in corresponding normal cells. II. COMPOSITIONS AND METHODS OF THE INVENTION

[0134] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of recombinant genetics, immunology, and biochemistry. Basic texts disclosing the general terms in molecular biology and genetics include e.g., Lackie, Dictionary of Cell and Molecular Biology, Elsevier (5th ed. 2013). Basic texts disclosing methods in recombinant genetics and molecular biology include e.g., Sambrook et al,Molecular Cloning— A Laboratory Manual, Cold Spring Harbor Press 4th Edition (Cold Spring Harbor, N.Y. 2012) and Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016). Basic texts disclosing the general methods and terms in biochemistry include e.g., Lehninger Principles of Biochemistry sixth edition, David L. Nelson and Michael M. Cox eds. W.H. Freeman (2012). Basic texts disclosing the general methods and terms immunology include Janeway's Immunobiology (Ninth Edition) by Kenneth M. Murphy and Casey Weaver (2017) Garland Science; Fundamental Immunology (Seventh Edition) by William E. Paul (2013) Lippincott, Williams and Wilkins. A. Chimeric Antigen Receptor Constructs:

[0135] The present disclosure provides a multispecific CAR T cell expressing two or more CARs, e.g., a dual CAR. In embodiments, in the T cell, the first CAR comprises a full length CD27 domain (for binding to CD70), and the other one or more CARs comprises one or more affinity binding domains that bind to one or more disease-associated antigens of interest. As demonstrated herein, the inclusion of full length CD27 for targeting CD70 with CAR T cells is superior to conventional anti-CD70 scFv approaches, as well as CD27 fragments, and provides multiple synergistic benefits. For example, multispecific CAR-T cells comprising full-length CD27 exhibit lower fratricide against CD70+ activated T cells and increased viability, and thus improved manufacturability. The multispecific CAR T cells of the subject invention also provide significant functional advantages compared to conventional anti-CD70 scFv-based approaches with improved anti-tumor activity, including against tumor cells with low CD70 expression.

[0136] In embodiments, a multispecific CAR T cell herein comprises one or more nucleic acids encoding the CARs. In some examples, the multispecific CAR T cell comprises multiple nucleic acids, each of which encodes one or more CARs. In some examples, the multispecific CAR T cell comprises a nucleic acid encoding two or more CARs. In such cases, the nucleic acid may further comprise one or more cleavage sequences between the CARs.

[0137] In embodiments, a nucleic acid encoding a dual CAR comprises from C-terminus to N- terminus, the first CAR targeting CD70, a cleavage sequence, and the second CAR targeting a disease-associated antigen. Alternatively, a nucleic acid encoding a dual CAR may comprise, from C-terminus to N-terminus, the second CAR targeting a disease-associated antigen, a cleavage sequence, and the first CAR targeting CD70.

[0138] In embodiments, the first CAR targets cells that exhibit cell surface expression of CD70. The cells that exhibit cell surface expression of CD70 can be activated immune cells (Bowman et al., J. Immunol. (1994);152:1756-1761; Nolte et al., Immunol. Rev. (2009);229:216- 231), stromal cells of the thymic medulla, and antigen-presenting cells (Keller et al., Proc Nat Acad Sci USA. (2007);104:5989–94; Borst et al., Curr Opin Immunol. (2005);17:275-81; Hendriks et al., J. Immunol. (2005);175:1665-76). The cells that exhibit cell surface expression, or overexpression, of CD70 can be hematological (Flieswasser et al., Cancers. (2019);11) and / or solid tumor cells (Pahl et al., Cancer Cell Int. (2015);15:31; Jilaveanu et al., Hum Pathol. (2012);43:1394–9; Hishima et al., Int J Cancer. (2002);98:352–6; Burvenich et al., Theranostics. (2018);8:4199–209).

[0139] In embodiments, cells that exhibit cell surface expression of a disease-associated antigen targeted by the second CAR include B cells at all phases of development including terminal differentiation into plasma cells such as, e.g. pro B cells, pre B cells, transitional / immature B cells, naive B cells, GC B cells, memory B cells, plasmablasts, and plasma cell. Cells that exhibit cell surface expression of a disease-associated antigen targeted by the second CAR may also include T cells such as Th1 / Th17 T cells, CD4+ T cells, keratinocytes, and cells described in Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat Rev Drug Discov.2021 Mar;20(3):179-199, which is incorporated by reference in its entirety.

[0140] In embodiments, the second CAR in a multispecific CAR T cell comprises one or more affinity binding domains that bind to one or more of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In embodiments, the antigen is a B cell antigen selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF- R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In embodiments, the antigen is a plasma cell antigen selected from the group comprising or consisting of CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB. In embodiments, the antigen is a T cell antigen selected from the group comprising or consisting of CD52, CD40L, alpha4 integrin, CXCR6, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L.

[0141] In embodiments, the second CAR in a multispecific T cell comprises an affinity binding domain that binds to CD19, e.., the CD19 binding domain in PL1605 as indicted in Table 1.

[0142] Additional examples of the affinity binding domains that bind to CD19 include those described in EP3214091, US10221245, US8906682, US10421810, US10639329, WO2010 / 052014, WO2015 / 109131, WO2017 / 134140, and WO 2020 / 018922, each of which is incorporated by reference herein in its entirety. Further examples of affinity binding domains that bind to CD19 include the CD19-binding domains in tisagenlecleucel, blinatumomab, tafasitamab, XmAb5574 (Xencor), AFM-11, inebilizumab, loncastuximab, MEDI-551 (Cellective Therapeutics), MDX-1342 (Medarex), A3B1, and FMC63-28Z.

[0143] In embodiments, the second CAR comprises an affinity binding domain that binds to FcRL5. Examples of the affinity binding domains that bind to FcRL5 include those described in WO2016205520, WO2014210064, WO2010114940, and WO2016090337, each of which is incorporated by reference in its entirety.

[0144] In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD20. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD22. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to BAFF-R. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to BCMA. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD38. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD138. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to FcRL5. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to GPRC50. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to TAC1. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD52. Inembodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to FcgRIIB. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD40. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD40L. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to IL-36. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to IL-36R. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to IL-6. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to IL-6R. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to alpha4 integrin. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CXCR6. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to DSG3. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to PD1. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to VISTA. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to BTLA. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to LAG3. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to ICOS. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targetingCD70) and a second CAR comprising an affinity binding domain that binds to ICOS-L. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD123. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD79b. In embodiments, the dual CAR construct herein encodes a first CAR comprising CD27 (for targeting CD70) and a second CAR comprising an affinity binding domain that binds to CD37.

[0145] Aspects of the invention include dual CARs, nucleic acids encoding dual CARs, and constructs and vectors containing such nucleic acids. In some cases, the nucleic acid is a, e.g., heterologous, component of an expression cassette. In embodiments, the nucleic acid is a, e.g., heterologous, component of a retroviral vector. In embodiments, the nucleic acid is a, e.g., heterologous, component of a T cell, e.g., an ^ ^ or γδ T cell, preferably a γδ T cell, and more preferably, a δ1 γδ T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of an γ+T cell and / or a δ+T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of an α- T cell and / or a β- T cell.

[0146] Described herein are nucleic acids encoding a CAR binding domain that specifically binds CD70. CD70 belongs to the TNF superfamily and has the amino acid sequence of SEQ ID NO: 1. CD70 is involved in the proliferation and survival of lymphoid-derived cells when it interacts with its receptor, CD27.

[0147] In the preferred embodiments described and exemplified herein, the first CAR in a dual CAR comprises both a CD27 antigen binding domain and a CD27 transmembrane domain. The CD27 amino acid sequence may comprise or consist of any suitable human antigen binding and transmembrane domain CD27 amino acid sequence. In the preferred embodiments, full-length CD27, including a signal peptide, antigen binding domain, transmembrane domain, and costimulatory signaling domain, has the amino acid sequence of SEQ ID NO: 32. In embodiments, the antigen binding domain of CD27 comprises or consists of amino acid residues 20-191 of SEQ ID NO: 32, and has the amino acid sequence of SEQ ID NO: 34, the transmembrane domain of CD27 comprises or consists of amino acid residues 192-212 of SEQ ID NO: 32, and has the amino acid sequence of SEQ ID NO: 36, and the costimulatory domain of CD27 is composed of amino acid residues 213-260 and has the amino acid sequence of SEQ ID NO: 38. Accordingly, in an embodiment of the invention, the CAR comprises an antigen binding – transmembrane domaincomprising the amino acid sequences of SEQ ID NOs: 34 and 36. The antigen binding domain of CD27 specifically binds to CD70.

[0148] In preferred embodiments, a nucleic acid encodes a CAR comprising a CD27 amino acid sequence composed of at least an antigen binding domain comprising or consisting of SEQ ID NO: 34, a transmembrane domain comprising or consisting of SEQ ID NO: 36, and a costimulatory domain comprising or consisting of SEQ ID NO: 38, and a signal peptide comprising or consisting of SEQ ID NO: 2.

[0149] In embodiments, the second CAR in a multispecific T cell comprises one or more affinity binding domains that binds to one or more autoimmune disease-associated antigens. Examples of antigens associated with autoimmune diseases include aggrecan, alanyl-tRNA syntetase (PL-12), alpha beta crystallin, alpha fodrin (Sptan 1), alpha-actinin, α1 antichymotrypsin, α1 antitripsin, α1 microglobulin, aldolase, aminoacyl-tRNA synthetase, an amyloid, an annexin, an apolipoprotein, aquaporin, bactericidal / permeability-increasing protein (BPI), β-globin precursor BP1, β-actin, β-lactoglobulin A, β-2-glycoprotein I, β2-microglobulin, a blood group antigen, C reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalase, cathepsin B, a centromere protein, chondroitin sulfate, chromatin, collagen, a complement component, cytochrome C, cytochrome P450 2D6, cytokeratins, decorin, dermatan sulfate, DNA topoisomerase I, elastin, Epstein-Barr nuclear antigen 1 (EBNA1), elastin, entaktin, an extractable nuclear antigen, Factor I, Factor P, Factor B, Factor D, Factor H, Factor X, fibrinogen, fibronectin, formiminotransferase cyclodeaminase (LC-1), gp210 nuclear envelope protein, GP2 (major zymogen granule membrane glycoprotein), a glutenin, glycoprotein gpIIb / IIIa, glial fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), haptoglobin A2, heat shock proteins, hemocyanin, heparin, a histone, histidyl-tRNA synthetase (Jo-1), a hordein, hyaluronidase, immunoglobulins, an integrin, interstitial retinol-binding protein 3, intrinsic factor, Ku (p70 / p80), lactate dehydrogenase, laminin, liver cytosol antigen type 1 (LC1), liver / kidney microsomal antigen 1 (LKM1), lysozyme, melanoma differentiation- associated protein 5 (MDAS), Mi-2 (chromodomain helicase DNA binding protein 4), a mitochondrial protein, muscarinic receptors, myelin-associated glycoprotein, myosin, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein, myeloperoxidase (MPO), rheumatoid factor (IgM anti-IgG), neuron-specific enolase, nicotinic acetylcholine receptor A chain, nucleolin, a nucleoporin, nucleosome antigen, PM / Sc1100, PM / Scl 75,pancreatic β-cell antigen, pepsinogen, peroxiredoxin 1, phosphoglucose isomerase, phospholipids, phosphatidyl inositol, platelet derived growth factors, polymerase beta (POLB), potassium channel KIR4.1, proliferating cell nuclear antigen (PCNA), proteinase-3, proteolipid protein, proteoglycan, prothrombin, recoverin, rhodopsin, ribonuclease, a ribonucleoprotein, ribosomes, a ribosomal phosphoprotein, RNA, an Sm protein, Sp100 nuclear protein, SRP54 (signal recognition particle 54 kDa), a selectin, smooth muscle proteins, sphingomyelin, streptococcal antigens, superoxide dismutase, synovial joint proteins, T1F1 gamma collagen, threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid stimulating hormone receptor, transferrin, triosephosphate isomerase, tubulin, tumor necrosis factor-alpha, topoisomerase, U1- dnRNP 68 / 70 kDa, U1-snRNP A, U1-snRNP C, U-snRNP B / B′, ubiquitin, vascular endothelial growth factor, vimentin, and vitronectin.

[0150] In embodiments, the second CAR in a multispecific T cell comprises one or more affinity binding domains that binds to one or more tumor-associated antigens. Examples of the tumor-associated antigen include CD19, CD20, CD30, CD22, CD33, CD37, CD38, CD56, CD79b, CD123, CD138, CD123, CD79b, CD75, CA6, GPRC50, TAC1, GD2, B7H6, B-cell maturation antigen (BCMA), B-cell activating factor receptor (BAFF-R), Fc Receptor-Like 5 (FcRL5), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-1, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c- Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, L1CAM, GPC2, GPC3, folate binding protein / receptor, SLC44A4, Cripto, CTAGIB, AXL, IL-13R, IL-3Rα2, SLTRK6, gp100, MART1, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3. MAGEA4), KKLC1, mutated ras, BRAF, p53, MHC class I chain-related molecule A (MICA), or MHC class I chain-related molecule B (MICB), and one or more antigens of HPV, CMV, or EBV.

[0151] In embodiments, the second CAR in a multispecific T cell comprises one or more affinity binding domains that binds to one or more pathogenic antigens. A pathogenic antigen may be a bacterial, viral, or fungal molecule, such as a bacterial, viral, or fungal protein. In embodiments, an antigen presenting cell may internalize pathogenic molecules (e.g., pathogenic proteins, nucleic acids, lipids, or fragments produced by a pathogenic organism such as a bacterium or a virus), for instance with phagocytosis or by receptor-mediated endocytosis, and display afragment of the antigen bound to an appropriate MHC molecule. For instance, various 9 mer fragments of a pathogenic protein may be displayed by an APC. Engineered, enriched immune cell populations of the disclosure may be designed to recognize various antigens and antigen fragments of a pathogenic bacterium or a virus.

[0152] Examples of pathogenic bacteria can be found in the: a) Bordetella genus, such as Bordetella pertussis species; b) Borrelia genus, such Borrelia burgdorferi species; c) Brucelia genus, such as Brucella abortus, Brucella canis, Brucela meliterisis, and / or Brucella suis species; d) Campylobacter genus, such as Campylobacter jejuni species; e) Chlamydia and Chlamydophila genuses, such as Chlamydia pneumonia, Chlamydia trachomatis, and / or Chlamydophila psittaci species; f) Clostridium genus, such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani species; g) Corynebacterium genus, such as Corynebacterium diphtheria species; h) Enterococcus genus, such as Enterococcus faecalis, and / or Enterococcus faecium species; i) Escherichia genus, such as Escherichia coli species; j) Francisella genus, such as Francisella tularensis species; k) Haemophilus genus, such as Haemophilus influenza species; 1) Helicobacter genus, such as Helicobacter pylori species; m) Legionella genus, such as Legionella pneumophila species; n) Leptospira genus, such as Leptospira interrogans species; o) Listeria genus, such as Listeria monocytogenes species; p) Mycobacterium genus, such as Mycobacterium leprae, mycobacterium tuberculosis, and / or mycobacterium ulcerans species; q) Mycoplasma genus, such as Mycoplasma pneumonia species; r) Neisseria genus, such as Neisseria gonorrhoeae and / or Neisseria meningitidia species; s) Pseudomonas genus, such as Pseudomonas aeruginosa species; t) Rickettsia genus, such as Rickettsia rickettsii species; u) Salmonella genus, such as Salmonella typhi and / or Salmonella typhimurium species; v) Shigella genus, such as Shigella sonnei species; w) Staphylococcus genus, such as Staphylococcus aureus, Staphylococcus epidermidis, and / or Staphylococcus saprophyticus spedes; x) Streptpcoccus genus, such as Streptococcus agalactiae, Streptococcus pneumonia, and / or Streptococcus pyogenes species; y) Treponema genus, such as Treponema pallidum species; z) Vibrio genus, such as Vibrio cholera; and / or aa) Yersinia genus, such as Yersinia pestis spedes.

[0153] Examples of pathogenic viruses can be found in the following families of viruses and are illustrated with exemplary species: a) Adenoviridae family, such as Adenovirus spedes; b) Herpesviridae family, such as Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus type 8 species; c)Papillomaviridae family, such as Human papillomavirus species; d) Polyomaviridae family, such as BK virus, JC virus species; e) Poxviridae family, such as Smallpox species; f) Hepadnaviridae family, such as Hepatitis B virus species; g) Parvoviridae family, such as Human bocavirus, Parvovirus B19 species; h) Astroviridae family, such as Human astrovirus spedes; i) Caliciviridae family, such as Norwalk virus species; j) Flaviviridae family, such as Hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae family, such as Rubella virus species; 1) Hepeviridae family, such as Hepatitis E virus species; m) Retroviiidae family, such as Human immunodeficiency virus (HIV) species; n) Orthomyxoviridaw family, such as Influenza virus species; o) Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabia virus species; p) Bunyaviridae family, such as Crimean-Congo hemorrhagic fever virus species; q) Filoviridae family, such as Ebola virus and / or Marburg virus spedes; Paramyxoviridae family, such as Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Hendra virus and / or Nipah virus spedes; r) Rhabdoviridae genus, such as Rabies virus species; s) Reoviridae family, such as Rotavirus, Orbivirus, Coltivirus and / or Banna virus species. In some examples, a virus is unassigned to a viral family, such as Hepatitis D.

[0154] In embodiments, the second CAR in a multispecific CAR T cell comprises one affinity biding domain. Alternatively, the second CAR can be a tandem CAR, e.g., a CAR comprising multiple affinity binding domains, that bind to different disease-associated antigens. In some examples, the affinity binding domain of the second CAR in a multispecific CAR T cell comprises a tandem sdAb, diabody, DART, sc-diabody, (scFv)2 / BITE, or a multivalent antibody or fragment described in Nuñez-Prado, et al., The coming of age of engineered multivalent antibodies, Drug Discov Today.2015 May;20(5):588-94.

[0155] In embodiments, the second CAR in a multispecific CAR T cell comprises affinity binding domains targeting CD19 and CD20, e.g., those described in Tong C et al., Optimized tandem CD19 / CD20 CAR-engineered T cells in refractory / relapsed B-cell lymphoma., Blood. 2020 Oct 1;136(14):1632-1644. In embodiments, the second CAR comprises affinity binding domains targeting FcRL5 and BCMA, e.g., those described in Jiang D et al., Chimeric antigen receptor T cells targeting FcRH5 provide robust tumour-specific responses in murine xenograft models of multiple myeloma., Nature Communications volume 14, Article number: 3642 (2023). In embodiments, the second CAR comprises affinity binding domains targeting CD19 and BCMA,e.g., those described in Shi M., Bispecific CAR T cell therapy targeting BCMA and CD19 in relapsed / refractory multiple myeloma: a phase I / II trial, Nat Commun.2024 Apr 20;15(1):3371. In embodiments, the second CAR comprises affinity binding domains targeting CD19 and BCMA, e.g., those described in Kang L et al., Characterization of novel dual tandem CD19 / BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma, Biomark Res.2020 May 13:8:14. In embodiments, the second CAR comprises affinity binding domains targeting CD38 and BCMA, e.g., those described in Feng Y. et al., Novel BCMA-OR-CD38 tandem-dual chimeric antigen receptor T cells robustly control multiple myeloma, Oncoimmunology, 2021 Aug 17;10(1):1959102. Further examples of tandem CARs include those CD19 / CD20 CARs, CD19 / CD22 CARs, CD19 / CD123 CARs, CD19 / CD79b CARs, CD19 / CD38 CARs, CD19 / CD37 CARs, CD19 / CD20 / CD22 CARs, e.g., those described in Furqan F. et al., Multispecific CAR T Cells Deprive Lymphomas of Escape via Antigen Loss, Annu Rev Med.2023 Jan 27:74:279-291.

[0156] In embodiments, a CAR encoded by a nucleic acid may further comprise at least one costimulatory domain, wherein the costimulatory domain comprises functional costimulatory signaling domain derived from e.g., a MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, and the like. For example, it is within the scope of this disclosure that the CAR can include 2, 3, 4 or more costimulatory domains. It is also within the scope of this disclosure that when more than one costimulatory domain is included, the costimulatory domains may be the same, or they may be different. In embodiments, the costimulatory domains are derived from one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3),SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, and TRANCE / RANKL, or a portion thereof, and combinations thereof.

[0157] In embodiments, a nucleic acid encoding a CAR encodes at least one 4-1BB costimulatory domain, and optionally a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two 4-1BB costimulatory domains, or at least two 4-1BB costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the 4-1BB costimulatory domain comprises SEQ ID NO: 4. In embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least one, at least two, or at least three or more modifications of an amino acid sequence of SEQ ID NO: 4. In embodiments, the 4-1BB costimulatory domain is substantially similar to the 4-1BB costimulatory domain comprising SEQ ID NO: 4.

[0158] In embodiments, a nucleic acid encoding a CAR encodes at least one CD27 costimulatory domain, and optionally at least one second costimulatory domain selected from 4- 1BB, ICOS, CD28, OX40, 2B4, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least one CD27 costimulatory domain, and a 4-IBB costimulatory domain. In embodiments, the nucleic acid encodes two CD27 costimulatory domains, and at least one second costimulatory domain selected from a 4-1BB, ICOS, CD28, and CD27. In embodiments, the CD27 costimulatory domain comprises SEQ ID NO: 38. In embodiments, the CD27 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 38. In embodiments, the CD27 costimulatory domain is substantially similar to the CD27 costimulatory domain comprising SEQ ID NO: 38.

[0159] In embodiments, a nucleic acid encoding a CAR encodes at least one CD28 costimulatory domain, and optionally a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two CD28 costimulatory domains, or at least two CD28 costimulatory domains in combination with one, two, three, or four,or more, costimulatory domains selected from a 4-1BB, ICOS, CD28, OX40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 64. In embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 65. Included in SEQ ID NO: 64 and SEQ ID NO: 65 are three subdomains YMNM, PRRP, and PYAP, that are capable to regulate signaling pathways. In embodiments, a disclosed CAR comprises mutation or deletion of one or more of said subdomains (see e.g., WO2019010383). In embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence of SEQ ID NO: 65. In embodiments, the CD28 costimulatory domain is substantially similar to the CD28 costimulatory domain comprising SEQ ID NO: 64. In embodiments, the CD28 costimulatory domain is substantially similar to the CD28 costimulatory domain comprising SEQ ID NO: 65.

[0160] In embodiments, a nucleic acid encoding a CAR encodes at least one ICOS costimulatory domain, and optionally a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two ICOS costimulatory domains, or at least two ICOS costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the ICOS costimulatory domain comprises SEQ ID NO: 66. In embodiments, the ICOS costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 66 (see e.g., US20170209492). In embodiments, the ICOS costimulatory domain is substantially similar to the ICOS costimulatory domain comprising SEQ ID NO: 66.

[0161] In embodiments, a nucleic acid encoding a CAR encodes at least one OX40 costimulatory domain, and optionally a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two OX40 costimulatory domains, or at least two OX40 costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the OX40 costimulatory domaincomprises SEQ ID NO: 67. In embodiments, the OX40 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 67. In embodiments, the OX40 costimulatory domain is substantially similar to the OX40 costimulatory domain comprising SEQ ID NO: 67.

[0162] In embodiments, a nucleic acid encoding a CAR encodes at least one intracellular signaling domain. In embodiments, the at least one intracellular signaling domain is additional to one or more costimulatory domains. In embodiments, the one or more intracellular signaling domains are included to increase proliferation, persistence, and / or cytotoxic activity of the host cell, preferably a γδ cell, harboring the CAR as herein disclosed. For example, in embodiments, the intracellular signaling domain(s) comprise CD3ζ, repeat (e.g., 2-5) DAP10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, and the like. It is within the scope of this disclosure that the endodomain of a disclosed CAR can include a plurality (e.g., 2, 3, 4, or more) of intracellular signaling domains. In a case where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same, or they may be different.

[0163] In embodiments, an intracellular signaling domain of a disclosed CAR is or comprises a CD3ζ signaling domain. In embodiments, a CD3ζ signaling domain is or comprises the amino acid sequence set forth in SEQ ID NO: 10, 12, or 13. In embodiments, the CD3ζ signaling domain those in PL1605 as indicated in Table 1.

[0164] In embodiments a nucleic acid encoding a CAR encodes one or more costimulatory domain(s) (e.g., 4-1BB costimulatory domain), and one or more intracellular signaling domain(s) (e.g., CD3ζ signaling domain). In embodiments, the nucleic acid encodes at least one 4-1BB costimulatory domain, at least one CD27 or CD28 domain, and at least one CD3ζ signaling domain. In other embodiments, the nucleic acid encodes one or more first costimulatory domains (e.g., 4-1BB, CD27, CD28, OX40, ICOS) and one or more second costimulatory domains (e.g., 4- 1BB, CD27, CD28, OX40, ICOS), and one or more intracellular signaling domains (e.g., CD3ζ). In embodiments, the CD3ζ signaling domain is downstream (C-terminal) to the costimulatory domain(s) (e.g., 4-1BB). In embodiments, the CD3ζ signaling domain is upstream (N-terminal) to the costimulatory domain(s) (e.g., 4-1BB).

[0165] In embodiments, a CAR other than the CAR targeting CD70 (e.g., the second CAR in a multispecific T cell herein) comprises a hinge domain and a transmembrane domain. For exampleand without limitation, a transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0166] In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is a transmembrane domain of CD8α. In certain embodiments, the transmembrane domain of CD8 comprises the amino acid sequence set forth in SEQ ID NO: 99. In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD28. In certain embodiments, the transmembrane domain of CD28 comprises the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the transmembrane domain comprises a transmembrane domain of ICOS. In certainembodiments, the transmembrane domain of ICOS comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0167] A hinge domain may be a hydrophilic region located between the affinity binding domain / means for specifically binding and the transmembrane domain. In embodiments, this domain facilitates proper protein folding for the CAR. The hinge domain is an optional component for the CAR. The hinge domain may include a domain selected from Fc fragments of antibodies, hinge domains of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. For example and without limitation, a hinge domain relevant to the present disclosure may be derived from the extracellular regions of type 1 membrane proteins such as CD8alpha, CD8beta, CD4, CD28, CD28T, 4-1BB, and CD7, which may be wild- type hinge domains from these molecules or may be altered. In embodiments, the hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or receptor-derived hinge. For example, hinge domains include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 124) and (GGGS)n (SEQ ID NO: 125), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge domains can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 127), GGSGG (SEQ ID NO: 128), GSGSG (SEQ ID NO: 129), GSGGG (SEQ ID NO: 130), GGGSG (SEQ ID NO: 131), GSSSG (SEQ ID NO: 132), and the like.

[0168] In embodiments, the hinge domain is an immunoglobulin heavy chain hinge domain. Immunoglobulin hinge domain amino acid sequences are known in the art; see, e.g., Tan et ah, Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et ah, Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge domain can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 105); CPPC (SEQ ID NO: 106); CPEPKSCDTPPPCPR (SEQ ID NO: 107) (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 108); KSCDKTHTCP (SEQ ID NO: 109); KCCVDCP (SEQ ID NO: 110); KYGPPCP (SEQ ID NO: 111); EPKSCDKTHTCPPCP (SEQ IDNO: 112) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 113) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 114) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 115) (human IgG4 hinge); and the like.

[0169] The hinge domain can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge domain. In one embodiment, the hinge domain can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge domain. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge domain comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 116); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897.

[0170] In certain embodiments, the hinge domain can comprise an amino acid sequence derived from human CD8, or a variant thereof. In certain embodiments, the CD8 alpha hinge sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 97. In certain embodiments, the hinge and transmembrane domain sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 101.

[0171] In embodiments, an isolated nucleic acid encoding one or more CARs herein can also encode for one or more multicistronic linker region(s) configured to facilitate translation of the CAR polypeptide(s) and one or more additional polypeptides. In embodiments, nucleic acids encoding the one or more additional polypeptides and associated linker region can be positioned at the 3’ end of the isolated nucleic acid, or at the 5’ end of the isolated nucleic acid, or in some examples at both the 5’ end and the 3’ end of the isolated nucleic acid.

[0172] In some examples, the linker region(s) can encode a self-cleavage and / or a cleavage sequence. In some examples, the self-cleavage sequence is a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A) which can induce ribosomal skipping during translation of the CAR. In embodiments, the cleavage sequence is a furin sequence. In some examples, the cleavage sequence (e.g., furin cleavage sequence as set forth in SEQ ID NO: 70) is amino terminal to a self-cleavage sequence, for example furin-P2A (FP2A). In embodiments, the multicistronic linker region encodes an internal ribosome entry site. In embodiments, the addition of an optional linker “GSG” or “SGSG” and the like can improve cleavage efficiency. In this way, the one or more additional polypeptides can be release from the CAR and directed to the secretory pathway.

[0173] In embodiments, the cleavage sequence is the FP2A amino acid sequence as set forth in SEQ ID NO: 6. In embodiments, the cleavage sequence is a P2A amino acid sequence as setforth in SEQ ID NO: 8, or SEQ ID NOs: 68-69. In embodiments, the cleavage sequence is a furin amino acid sequence as set forth in SEQ ID NO: 70. In embodiments, the cleavage sequence is a F2A amino acid sequence as set forth in SEQ ID NO: 71. In embodiments, the cleavage sequence is a E2A amino acid sequence as set forth in SEQ ID NO: 72. In embodiments, the cleavage sequence is a T2A amino acid sequence as set forth in SEQ ID NO: 73. Additional cleavage sequences of interest can be found in de Lima and Lanza, “2A and 2A-like Sequences.” Viruses (2021) 13:112160.

[0174] In certain aspects, multiple cleavage and / or self-cleavage sequences can be encoded carboxy-terminal to signaling and / or costimulatory domain(s) and amino-terminal to an encoded one or more additional polypeptide. In certain aspects, one or more self-cleavage sequences and one or more sequences cleaved by an endogenous protease are encoded in a construct described herein. In certain embodiments, an endogenous protease recognition site is encoded amino terminal to a self-cleavage sequence. Further suitable internal ribosome entry sites include, but are not limited to, those disclosed e.g., in Nucleic Acids Res.2010 Jan;38(Database issue):D131- 6. doi: 10.1093 / nar / gkp981. Epub 2009 Nov 16, those described at iresite.org, those described in WO 2018 / 215787, the sequence described in GenBank accession No. KP019382.1, and the IRES element disclosed in GenBank accession No. LT727339.1. Additional multi-cistronic linker regions, including cleavage self-cleavage, and IRES elements, are disclosed in US 2018 / 0360992 and U.S.8,865,467.

[0175] In embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by the nucleotide sequence set forth in SEQ ID NO: 74. Another exemplary internal ribosome entry site is encoded by the nucleotide sequence set forth in SEQ ID NO: 75.

[0176] In embodiments, the one or more additional polypeptides include one or more soluble gamma chain cytokines expressed as separate polypeptides from the CAR. The one or more soluble common gamma chain cytokines can include but are not limited to IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In embodiments, the common gamma chain cytokine is IL-15. IL-15 sequences, including codon optimized nucleic acid sequences encoding soluble IL-15 (sIL-15) are disclosed herein and in WO 2007 / 037780.

[0177] In embodiments, the one or more additional polypeptides include one or more labels or markers, for example to facilitate an ability to monitor CAR expression level, serve as an internal control, and the like. In embodiments, an isolated nucleic acid encoding a CAR encodes for a fluorescent protein, examples of which include but are not limited to green fluorescent protein (GFP), red fluorescent protein (RFP), enhanced GFP (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), and the like. Other examples can include but are not limited to chloramphenicol acetyltransferase, beta-galactosidase, beta-glucuronidase, beta-lactamase, luciferase, and the like.

[0178] In embodiments, the one or more additional polypeptides include a protein that is expressed on a cell surface to facilitate detection and / or isolation of cells expressing said protein, e.g., via fluorescent activated cell sorting (FACS); or for enrichment through positive selection using an antibody specific to the encoded protein, e.g., use of an antibody to purify or enrich the cells product on a column or apparatus; or for in vivo binding of an antibody to the protein to enhance or eliminate activity, e.g., to facilitate removal of cells expressing the protein in patients as a safety consideration. Exemplary proteins useful for these purposes include, e.g., CD19, CD20 (Rituxumab recognition domain), LNGFR, a truncated form of the human epidermal growth factor receptor (EGFRt), and the like. By way of example, EGFRt can be targeted by a clinical stage antibody, where such treatment of a patient with said antibody results in elimination of cells containing an isolated nucleic acid encoding a CAR and / or said CAR as disclosed herein.

[0179] In embodiments, the one or more additional polypeptides include a protein that functions to increase resistance to exhaustion and activation-induced apoptosis and / or upregulate one or more proinflammatory cytokines, costimulatory molecules and / or antigen presentation machinery. A representative example includes but is not limited to lymphotoxin beta receptor (LTBR). LTBR is typically expressed in a subset of myeloid cells but is absent in lymphocytes. When expressed in T cells, LTBR may induce transcriptional remodeling that imparts the T cell with one or more of the above-mentioned advantageous functions (Legut et al., Blood. (2021); 138(1): 1726).

[0180] In embodiments, the one or more additional polypeptides includes a polypeptide that imparts host cells with the capability to resist tumor antigen-specific cellular immunity, for example that mediated by transforming growth factor beta (TGF-β). For example, an isolated nucleic acid may encode a dominant negative receptor for TGF-beta (dnTGFβR2), e.g., asdescribed in Foster et al., J Immunother. (2008); 31: 500-505, WO2019 / 173324A1, WO2020 / 183131A1, and WO2020042647A1. Incorporation of such a dominant negative receptor for TGF-beta may provide a functional advantage over control cells that lack such a dominant negative receptor for TGF-beta in the presence of a TGF-beta-secreting tumor, including enhanced anti-tumor activity. In embodiments, an isolated nucleic acid encodes a signal peptide operably linked to facilitate directing of the one or more additional polypeptides to the secretory pathway. Such one or more additional polypeptides can be those that reside inside certain organelles, are secreted from the host cell, or are inserted into cellular membranes. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 2. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 16. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 20. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 24. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 28. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 76.

[0181] In embodiments, the one or more additional polypeptides includes the EGFRt amino acid sequence as set forth in SEQ ID NO: 18. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 16 is operably linked to SEQ ID NO: 18. In embodiments, the one or more additional polypeptides includes the dominant-negative TGFβ receptor II (dnTGFβR2) amino acid sequence as set forth in SEQ ID NO: 22. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 20 is operably linked to SEQ ID NO: 22. In embodiments, the one or more additional polypeptides includes the full-length LTBR amino acid sequence as set forth in SEQ ID NO: 26. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 24 is operably linked to SEQ ID NO: 26. In embodiments, the one or more additional polypeptides includes the LNGFR amino acid sequence as set forth in SEQ ID NO: 30. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 28 is operably linked to SEQ ID NO: 30. In embodiments, the one or more additional polypeptides includes the sIL-15 amino acid sequence as set forth in SEQ ID NO: 77. In embodiments, the signal peptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 76 is operably linked to SEQ ID NO: 77.

[0182] In embodiments, the one or more additional polypeptides includes a chimeric switch receptor comprising an extracellular domain of a TGFβ receptor for binding to TGFβ (e.g., TGFβRI and / or TGFβRII), and an intracellular domain of a cytokine receptor. The chimeric switch receptors can convert a TGFβ signal into a cytokine signal that promotes cytotoxicity. Examples of such chimeric switch receptors include those descried in WO2012138858, WO2016122738, WO2018094244, WO2014172584, WO2019109980, and WO2022037562, each of which is incorporated by reference in its entirety.

[0183] In embodiments, the one or more additional polypeptides includes a dominant negative Fas (dnFas). Incorporation of such a dominant negative Fas in a T cell may provide a functional advantage over control cells that lack such a dominant negative Fas in the prevention of Fas ligand- induced apoptosis and allowing for T cell persistence and antitumor efficacy. Examples of the dnFas include that described in Yamamoto TN et al., T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy, J Clin Invest.2019 Feb 25;129(4):1551- 1565, which is incorporated by reference herein in its entirety.

[0184] In embodiments, the one or more additional polypeptides includes a membrane-bound IL-12 (mbIL-12). Incorporation of such a mbIL-12 in a T cell may provide a functional advantage over control cells that lack such a mbIL-12 in enhancing effector functions of the T cells and / or limiting the systemic toxicity associated with IL-12. Examples of the mbIL-12 include those described in Hu J. et al., Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors, J Immunother Cancer. 2022 Jan;10(1):e003633; Hombach A. et al., IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors, Mol Ther. 2022 Feb 2;30(2):593-605; and Lee EH et al., Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting, bioRxiv.2023 Jan 7;2023.01.06.522784, each of which is incorporated by reference herein in its entirety.

[0185] In embodiments, the one or more additional polypeptides an exogenous stem cell factor (SCF). The term “stem cell factor (SCF)” refers to a cytokine that that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Endogenous SCF is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. It is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a role in mast cell development, gametogenesis, andmelanogenesis. SCF through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions (see e.g., Mazzoldi, E.L., et al. A juxtacrine / paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer. Cell Death Dis 10, 412 (2019)). In embodiments, SCF includes a soluble form SCF (e.g., comprising or consisting of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, (human SCF based on NP_000890, first 165 aa of the mature peptide without signal peptide), SEQ ID NO: 134). In embodiments, SCF includes a transmembrane form SCF (e.g., comprising or consisting of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRNDSSSSNRKAKN PPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEK EREFQEV (human SCF based on NP_000890, mature peptide without signal peptide. SEQ ID NO:135), see also Aderson DM et al. Cell Growth Differ.1991 Aug;2(8):373-8), or comprising or consists of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGA LYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (human SCF based on NP_ NP_003985, mature peptide without signal peptide, SEQ ID NO: 136), see also Johan Lennartsson et al. Physiol Rev.2012 Oct;92(4):1619-49). Examples of soluble and transmembrane forms of SCF also include those described in J G Flanagan et al., Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant Cell.1991 Mar 8;64(5):1025-35; DM Anderson et al., Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24, Cell Growth Differ.1991 Aug;2(8):373-8; and Johan Lennartsson et al., Stem cell factor receptor / c-Kit: from basic science to clinical implications, Physiol Rev.2012 Oct;92(4):1619-49, each of which is incorporated by reference in its entirety.

[0186] In embodiments, the exogenous SCF may be a recombinant or synthetic stem cell factor, such as ancestim, a 166-amino-acid protein produced by E. coli bacteria into which a gene has been inserted for soluble human stem cell factor (MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSL TDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIF NRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, SEQ ID NO: 137) as described in McNiece IK and Briddel RA, The Cytokine Handbook (Fourth Edition), 2003); and MG da Silva et al. Ancestim (recombinant human stem cell factor, SCF) in association with filgrastim does not enhance chemotherapy and / or growth factor-induced peripheral blood progenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection, Bone Marrow Transplant.2004 Oct;34(8):683-91, each of which is incorporated by reference herein in its entirety.

[0187] Additional examples of recombinant and synthetic stem cell factor molecules include those described in WO1991005795 (titled “Stem cell factor”); US6020469 (titled “Stem cell factor formulations and methods”); US6759215 (titled “Method of preparing human stem cell factor polypeptide”); and US6218148 (titled “DNS encoding stem cell factor”), each of which is incorporated by reference herein in its entirety. In embodiments, the exogenous SCF is a SCF analog peptide, e.g., those described in US5885962 (titled “Stem cell factor analog compositions and method”); and Tilayov T. et al. Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies, Molecules. 2020 Oct 21;25(20):4850, each of which incorporated herein in its entirety.

[0188] The present disclosure provides CARs with “substantial identity” or “substantial similarity” to the sequences provided herein. The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), there is nucleotide sequence identity in %, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0189] As applied to polypeptides, the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity. In some aspects, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol.24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 144345, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0190] Sequence identity and / or similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Sequences also can be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol.215: 403-410 and (1997) Nucleic Acids Res.25:3389-3402, each of which is herein incorporated by reference.

[0191] Alternatively or additionally, a parent CAR of the present disclosure can comprise at least one non-conservative amino acid substitution. In this case, it is preferable for the non- conservative amino acid substitution to not interfere with or inhibit the biological activity of the parent CAR. The non-conservative amino acid substitution may enhance the biological activity of the modified CAR, such that the biological activity of the modified CAR is increased as compared to the parent CAR.

[0192] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 42, a CAR polypeptide PL648 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, an FP2A cleavage domain, and an EGFP domain.

[0193] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 44, a CAR polypeptide PL1090 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, an FP2A cleavage domain, a signal peptide, and an EGFRt domain.

[0194] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 46, a CAR polypeptide PL1093 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, an FP2A cleavage domain, a signal peptide, and a dnTGFβR2 domain.

[0195] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 48, a CAR polypeptide PL1106 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, an FP2A cleavage domain, a signal peptide, and a LTBR domain.

[0196] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 50, a CAR polypeptide PL1107 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0197] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 52, a CAR polypeptide PL1110 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, an FP2A signaling domain, and an LNGFRt domain.

[0198] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 54 a CAR polypeptide PL1179 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, a P2A cleavage domain, a signal peptide, and a dnTGFβR2 domain.

[0199] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 56, a CAR polypeptide PL1099 comprising the following domains in order: a full-length CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, a P2A cleavage domain, a signal peptide, and a LNGFRt domain.

[0200] In any of the above-mentioned CAR polypeptides encoded by said isolated nucleic acids, a FP2A cleavage domain may be substituted by a P2A cleavage domain (or other cleavage domain), and vice versa.

[0201] In embodiments, a nucleic acid encoding a PL648 CAR comprises the sequence of SEQ ID NO: 43.

[0202] In embodiments, a nucleic acid encoding a PL1090 CAR comprises the sequence of SEQ ID NO: 45.

[0203] In embodiments, a nucleic acid encoding a PL1093 CAR comprises the sequence of SEQ ID NO: 47.

[0204] In embodiments, a nucleic acid encoding a PL1106 CAR comprises the sequence of SEQ ID NO: 49.

[0205] In embodiments, a nucleic acid encoding a PL1107 CAR comprises the sequence of SEQ ID NO: 51.

[0206] In embodiments, a nucleic acid encoding a PL1110 CAR comprises the sequence of SEQ ID NO: 53.

[0207] In embodiments, a nucleic acid encoding a PL1179 CAR comprises the sequence of SEQ ID NO: 55.

[0208] In embodiments, a nucleic acid encoding a PL1099 CAR comprises the sequence of SEQ ID NO: 57.

[0209] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 58, a CAR polypeptide PL1091 comprising the following domains in order: a truncated CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, a FP2A cleavage domain, and an EGFRt domain.

[0210] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 60, a CAR polypeptide PL1094 comprising the following domains in order: a truncated CD27 domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, a FP2A cleavage domain, and an EGFP domain.

[0211] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 62, a CAR polypeptide PL1108 comprising the following domains in order: a truncated CD27 domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0212] In any of the above-mentioned CAR polypeptides comprising a truncated CD27, an FP2A cleavage domain may be substituted by a P2A cleavage domain (or other cleavage domain). Additionally or alternatively, another additional polypeptide, for example dnTGFβR2, may be substituted for EGFP, or EGFRt.

[0213] In embodiments, a nucleic acid encoding a PL1091 CAR comprises the sequence of SEQ ID NO: 59.

[0214] In embodiments, a nucleic acid encoding a PL1094 CAR comprises the sequence of SEQ ID NO: 61.

[0215] In embodiments, a nucleic acid encoding a PL1108 CAR comprises the sequence of SEQ ID NO: 63.

[0216] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 118, a polypeptide PL1065 comprising two CARs with the following domains in order: a signal peptide, a full-length CD27 domain, a first 4-1BB costimulatory domain, a first CD3ζ signaling domain, a P2A cleavage domain, a signal peptide, an anti-CD19 scFv, CD8α hinge and transmembrane domains, a second4-1BB costimulatory domain, and a second CD3ζ signaling domain. In embodiments, a nucleic acid encoding a PL1065 CAR comprises the sequence of SEQ ID NO: 117. The recombinant proteins from PL1605 post cleavage comprise or consist of the amino acid sequences set forth in SEQ ID NOS: 138 and 139.

[0217] The domain positions in PL1605 are shown in Table 1 below. Table 1. PL1605: pSINBA-flCD27-41BB-CD3z-P2A-Kymriah-CTL019.CO Annotation Numbering Full length CD27 1-780 Signal Peptide 1-57 Extracellular Domain 58-573 Transmembrane Domain 574-636 Intracellular Domain 637-780 4-1BB Co-Stimulatory Domain 781-906 CD3 Zeta Signaling Domain 907-1245 P2A 1246-1311 Kymriah-CTL019 1312-2769 Signal Peptide 1312-1374 Light chain variable 1375-2100 GS Linker 1696-1740 Heavy chain variable 1741-2100 CD8 Hinge and Transmembrane 2101-2307 Domain 4-1BB Co-Stimulatory Domain 2308-2433 CD3 Zeta Signaling Domain 2434-2769

[0218] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 120, a CAR polypeptide PL1026 comprising two CARs with following domains in order: a signal peptide, an anti-CD19 scFv, CD8α hinge and transmembrane domains, a first 4-1BB costimulatory domain, a first CD3ζ signaling domain, a P2A cleavage domain, a signal peptide, a full-length CD27 domain, a second 4-1BB costimulatory domain, a second CD3ζ signaling domain, and a P2A tag. In embodiments, a nucleic acid encoding a PL1026 CAR comprises the sequence of SEQ ID NO: 119. The recombinant proteins from PL1626 post cleavage comprise or consist of the amino acid sequences set forth in SEQ ID NOS: 140 and 141.

[0219] The domain positions in PL1626 are shown in Table 2 below. Table 2. PL1626: pSINBA-Kymriah-CTL019.CO-FP2A-flCD27-41BB-CD3z-P2A(P22Δ) Annotation Numbering Kymriah CTL019 1-1458 Signal Peptide 1-63 Light chain variable 64-384 GS Linker 385-429 Heavy chain variable 430-789 CD8 Hinge and Transmembrane 790-996 Domain 4-1BB Co-Stimulatory Domain 997-1122 CD3 Zeta Signaling Domain 1123-1458 Furin-P2A 1459-1539 Full length CD27 1540-2319 Signal Peptide 1540-1596 Extracellular Domain 1597-2112 Transmembrane Domain 2113-2175 Intracellular Domain 2176-2319 4-1BB Co-Stimulatory Domain 2320-2445 CD3 Zeta Signaling Domain 2446-2784 P2A(P22Δ) 2785-2847

[0220] In embodiments, an isolated nucleic acid encodes SEQ ID NO: 122, a CAR polypeptide PL1027 comprising two CARs with following domains in order: a signal peptide, an anti-CD19 scFv, CD8α hinge and transmembrane domains, a first 4-1BB costimulatory domain, a first CD3ζ signaling domain, a P2A cleavage domain, a signal peptide, a full-length CD27 domain, a second 4-1BB costimulatory domain, a second CD3ζ signaling domain, and a T2A tag. In embodiments, a nucleic acid encoding a PL1027 CAR comprises the sequence of SEQ ID NO: 121. The recombinant proteins from PL1627 post cleavage comprise or consist of the amino acid sequences set forth in SEQ ID NOS: 142 and 143.

[0221] The domain positions in PL1627 are shown in Table 3 below. Table 3. PL1627: pSINBA-Kymriah-CTL019.CO-FP2A-flCD27-41BB-CD3z-T2A(P21Δ)Annotation Numbering Kymriah CTL019 1-1458 Signal Peptide 1-63 Light chain variable 64-384 GS Linker 385-429 Heavy chain variable 430-789 CD8 Hinge and Transmembrane 790-996 Domain 4-1BB Co-Stimulatory Domain 997-1122 CD3 Zeta Signaling Domain 1123-1458 Furin-P2A 1459-1539 Full length CD27 1540-1596 Signal Peptide 1540-2319 Extracellular Domain 1597-2112 Transmembrane Domain 2113-2175 Intracellular Domain 2176-2319 4-1BB Co-Stimulatory Domain 2320-2445 CD3 Zeta Signaling Domain 2446-2784 T2A(P21Δ) 2785-2844

[0222] Representative CARs of the present disclosure are summarized in Table 4 along with associated SEQ ID NOs corresponding to CAR amino acid sequence. Additional sequences relevant to the present disclosure are summarized in Table 4 below. Table 4. Description of CARs (amino acid) PL648 (SEQ ID NO: 42) Full-length CD27, 41BB, CD3z - FP2A - EGFP PL1090 (SEQ ID NO: 44) Full-length CD27, 41BB, CD3z - FP2A - EGFRt PL1093 (SEQ ID NO: 46) Full-length CD27, 41BB, CD3z - FP2A - dnTGFBR2 PL1106 (SEQ ID NO: 48) Full-length CD27, 41BB, CD3z - FP2A - LTBR (Tumor necrosis factor receptor superfamily member 3)PL1107 (SEQ ID NO: 50 Full-length CD27, 41BB, CD3z PL1110 (SEQ ID NO: 52) Full-length CD27, 41BB, CD3z-FP2A-LNGFRt PL1179 (SEQ ID NO: 54) Full-length CD27, 41BB, CD3z - P2A - dnTGFBR2 PL1099 (SEQ ID NO: 56) Full-length CD27, 4-1BB, CD3z - P2A - LNGFRt PL1091 (SEQ ID NO: 58) Truncated CD27, 41BB, CD3z - FP2A - EGFRt PL1094 (SEQ ID NO: 60) Truncated CD27, 41BB, CD3z - FP2A - EGFP PL1108 (SEQ ID NO: 62) Truncated CD27, 41BB, CD3z PL1605 (SEQ ID NO: 118) Full-length CD27, 41BB, ADI CD3z , P2A, FMC63 scFv, CD8 hinge, CD8 TM, 4-1BB, Kym CD3z. More detailed domain structure is shown in FIG.26A. PL1626 (SEQ ID NO: 120) FMC63 scFv, CD8 hinge, CD8 TM, 4-1BB, Kym CD3z, P2A, full- length CD27, 4-1BB, ADI CD3z, P2A tag. More detailed domain structure is shown in FIG.26B. PL1627 (SEQ ID NO: 122) FMC63 scFv, CD8 hinge, CD8 TM, 4-1BB, Kym CD3z, P2A, full- length CD27, 4-1BB, ADI CD3z, T2A tag. More detailed domain structure is shown in FIG.26B. B. Expression of CAR constructs

[0223] As used herein, an isolated nucleic acid is intended to mean a DNA molecule which can be transformed or introduced into a host cell, such as a T cell, preferably a γδ T cell, more preferably a δ1 γδ T cell, and be transcribed and translated to produce a product (e.g., a dualCAR as herein described). In the isolated nucleic acids, a promoter is operably linked to the nucleic acid sequence encoding the CAR(s), i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA encoding the CAR(s). The term “operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.

[0224] The promoter can be of genomic origin or synthetically generated. A variety of promoters for use in host cells relevant to the present disclosure are well-known in the art (e.g., the CD4 promoter disclosed by Marodon, et al. (2003) Blood 101(9):3416-23). The promoter can be constitutive or inducible, where induction is associated with the specific cell type or a specific level of maturation. Alternatively, a number of well-known viral promoters may also be suitable. Promoters of interest include the β-actin promoter, SV40 early and late promoters, immunoglobulin promoter, human cytomegalovirus promoter, retrovirus promoter, and the Friend spleen focus-forming virus promoter. The promoters may or may not be associated with enhancers, wherein the enhancers may be naturally associated with the particular promoter or associated with a different promoter. In embodiments, expression of a CAR polypeptide is under the control of an inducible promoter, for example a promoter that is inducible by a molecule present in a disease microenvironment (e.g., TGFβ).

[0225] The sequence of the open reading frame encoding the various segments of the CARs of the present disclosure can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof.

[0226] In embodiments, for expression of one or more CARs, the naturally occurring or endogenous transcriptional initiation region of the nucleic acid sequence herein can be used to generate the CAR(s) in the host cell. Alternatively, an exogenous transcriptional initiation region can be used which allows for constitutive or inducible expression, wherein expression can optionally be controlled depending upon the host cell, the level of expression desired, the nature of the host cell, and the like.

[0227] A termination region encoding a C-terminal component of the CAR(s) can be included. Generally speaking, the source of the termination region is not considered to be critical to the expression of a recombinant protein and a wide variety of termination regions can be employed without adversely affecting expression.

[0228] The isolated nucleic acid, which encodes the CAR(s) can be prepared in conventional ways. Sequences (natural or synthetic) are isolated and manipulated, as appropriate, so as to allow for the proper joining of the various components. Thus, various nucleic acid sequences encoding for the various segments of the CAR(s) can be isolated, e.g., by employing the polymerase chain reaction (PCR), using appropriate primers. If necessary, specific primers can be designed which result in deletion of undesired portions of a nucleic acid sequence being used as a template.Additionally or alternatively, restriction digests of cloned genes can be used to generate the isolated nucleic acid constructs of the present disclosure. In either case, the sequences can be selected to provide for restriction sites which are blunt-ended, or have complementary overlaps for facilitating incorporation into various vectors. In examples, modification of a nucleic acid sequence (e.g., to introduce one or more point mutations, insertions or deletions), is performed. Methods for introducing modifications into nucleic acid sequences are known in the art and can include the use of various kits available for purchase (e.g., QuickChange Site Directed Mutagenesis Kit, Agilent, Santa Clara, CA).

[0229] The various manipulations for preparing the isolated nucleic acid encoding the CAR(s) of the present disclosure can be carried out in vitro. In embodiments a sequence encoding the CAR(s) is introduced into vectors for cloning and expression in an appropriate host cell using standard transformation or transfection methods. Thus, after each manipulation, the resulting construct from joining of the DNA sequences is cloned, the vector isolated, and the sequence screened to ensure that the sequence encodes the desired CAR(s). The sequence can be screened by restriction analysis, sequencing, or the like.

[0230] It is contemplated that the isolated nucleic acid can be introduced into host cells as naked DNA or in a suitable vector. Many suitable vectors are known to those skilled in molecular biology, the choice of which would depend on the function desired and include plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering. Methods that are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook et al. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the disclosure can be reconstituted into liposomes for delivery to target cells.

[0231] Methods of stably transfecting host cells by electroporation using naked DNA are known in the art (see for example U.S. Pat. No.6,410,319 disclosing T cell transfection). Naked DNA generally refers to the DNA encoding a CAR herein contained in a plasmid expression vector in proper orientation for expression. Advantageously, the use of naked DNA reduces the time required to produce host cells expressing CARs.

[0232] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector) can be used to introduce the isolated nucleic acidencoding a CAR into host cells. Suitable vectors for use in accordance with the method are non- replicating in the T-cells. A large number of vectors are known which are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Illustrative vectors include the pFB-neo vectors (STRATAGENE®) as well as vectors based on HIV, SV40, EBV, HSV or BPV.

[0233] Thus, it may be understood that in embodiments, the isolated nucleic acid is a circular nucleic acid. In embodiments, the isolated nucleic acid is a vector, such as a plasmid vector, an adenoviral vector, an adeno-associated viral vector, a viral vector, a retroviral vector, (e.g., a gamma retroviral vector), or a lentiviral vector. In embodiments, the isolated nucleic acid, or an, e.g., contiguous, portion thereof is integrated into the genome of a host cell, such as a host ^ ^ ^T cell. In an exemplary embodiment, the isolated nucleic acid is retroviral vector. C. T Cells

[0234] In embodiments, multispecific CAR T cells comprising two or more CARs herein have in vitro or in vivo cytotoxic activity against a disease-associated cell, e.g., an autoimmune disease- associated cell (e.g., autoimmune disease-associated B cell, plasmablast, or T cell) or a tumor cell (e.g., hematological and / or solid tumor cell), that exhibits cell surface expression of CD70 and / or another disease-associated antigen.

[0235] In some cases, the cytotoxic activity is innate activity. In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of CAR constructs having binding domains that specifically bind CD70 and another disease-associated antigen expressed on the surface of a disease-associated cell. In some cases, the T cells exhibit disease- associated cell killing activity greater than an innate level of in vitro and / or in vivo disease- associated cell killing activity in a control T cell.

[0236] In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of CAR constructs having binding domains that specifically bind CD70 and another disease-associated antigen expressed on the surface of an autoimmune disease- associated cell (e.g., autoimmune disease-associated B cell, plasmablast, or T cell). In some cases, the T cells exhibit autoimmune disease-associated cell killing activity greater than an innate level of in vitro and / or in vivo autoimmune disease-associated cell killing activity in a control T cell.

[0237] In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of the CAR constructs having binding domains that specifically bindCD70 and another disease-associated antigen expressed on the surface of a hematological and / or solid tumor cell. In some cases, the T cells exhibit hematological and / or solid tumor cell killing activity greater than an innate level of in vitro and / or in vivo hematological and / or solid tumor cell killing activity in a control T cell.

[0238] In some cases, the control T cell does not comprise a CAR construct. In some cases, the control T cell comprises a CAR construct lacking one or more of a binding domain described herein, a hinge domain described herein, a transmembrane domain described herein, an intracellular signaling domain described herein, and / or a costimulatory domain described herein.

[0239] In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct disclosed herein. In some cases, the T cells functionally express the CARs disclosed herein encoded by an isolated nucleic acid described herein.

[0240] The T cells described herein can exhibit robust and / or persistent disease-associated cell (e.g., autoimmune disease-associated cell or hematological and / or solid tumor cell) killing activity. In some cases, the disease-associated cell killing activity can persist for at least about 6 days to about 120 days, or for at least about 6 days to about 180 days, from first contact with a disease- associated cell. In some cases, the disease-associated cell killing activity of a T cell described herein, or a progeny thereof, can persist for at least about 6 days to about 120 days, or for at least about 6 days to about 180 days, from first contact with a disease-associated cell, or from administration of the T cell described herein. This persistent disease-associated cell killing activity can be exhibited in vitro, in vivo, or both in vitro and in vivo.

[0241] In some cases, the proliferation is an innate activity. In some cases, the proliferation is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct herein. In some cases, the T cells exhibit a greater level of in vitro and / or in vivo proliferation as compared to a control T cell. In some cases, the control T cell does not comprise a CAR construct. In some cases, the control T cell comprises one or more CAR constructs lacking a binding domain described herein, a hinge domain described herein, a transmembrane domain described herein, an intracellular signaling domain described herein, and / or a costimulatory domain described herein.

[0242] In some cases, the proliferation is at least in part, significantly (> about 20 or > about 25%), or entirely, due to the presence of the CARs herein. In some cases, T cells expressing CARsherein exhibits proliferation in response to contact with a cell, for example a disease-associated cell such as autoimmune disease-associated cell or a hematological and / or solid tumor cell that exhibits cell surface expression of CD70 and / or another disease-associated antigen.

[0243] T cells described herein can exhibit robust and / or persistent proliferation in a host organism that comprises a cell, for example a disease-associated cell that exhibits cell surface expression, or overexpression, of CD70 and / or another disease-associated antigen. In some cases, the proliferation can persist for at least about 6 days to about 120 days, or for at least about 6 days to about 180 days, from first contact with a cell expressing CD70 and / or another disease-associated antigen, for example a disease-associated cell such as an autoimmune disease-associated cell or a hematological and / or solid tumor cell, or from a date of administration of the T cell to the host organism. In some cases, the proliferation of a T cell described herein, or a progeny thereof, in the host organism that comprises the a disease-associated cell such as autoimmune disease- associated cell or hematological and / or solid tumor cell that exhibits cell surface expression, or overexpression, of CD70 and / or another disease-associated antigen can persist for at least about 6 days to about 120 days, or for at least about 6 days to about 180 days, from first contact with a cell expressing CD70 and / or another disease-associated antigen, for example a disease-associated cell such as an autoimmune disease-associated cell or a hematological and / or solid tumor cell, or from the date of first administration of the T cell to the host organism. In some cases, the proliferation in the host organism is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of the CAR constructs herein.

[0244] In embodiments, the T cells described herein express, or persistently express, pro- inflammatory cytokines such as, but not limited to, tumor necrosis factor alpha or interferon gamma after contact with a cell expressing CD70 and / or another disease-associated antigen, for example a disease-associated cell such as an autoimmune disease-associated cell or a hematological and / or solid tumor cell. In embodiments, the T cells described herein, or progeny thereof, express, or persistently express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with the cell expressing CD70 and / or another disease- associated antigen, e.g., in a host organism comprising a cell expressing CD70 and / or another disease-associated antigen such as an autoimmune disease-associated cell or hematological and / or solid tumor cell.γδ T cells

[0245] In embodiments, the T cell herein is a γδ T cell, e.g., a δ1 cell. The γδ T cells described herein can be δ1, δ2, δ3, or δ4 γδ T cells, or combinations thereof. In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ2- γδ T cells. In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ1 γδ T cells. In embodiments, the γδ T cells are δ1 γδ T cells.

[0246] In embodiments, provided herein is a cell population that comprises at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 γδ T cells. In embodiments, the cell population comprises at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1 or δ2 γδ T cells. In embodiments, the cell population comprises at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ2- γδ T cells. In embodiments, the cell population comprises at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1 γδ T cells. Modifications of T cells

[0247] In embodiments, T cells described herein can be modified to comprise one or more gene edits. Discussed herein, gene editing is a type of genetic engineering in which nucleotide(s) / nucleic acid(s) is / are inserted, deleted, and / or substituted in a DNA sequence, such as the genome of a T cell. Targeted gene editing enables insertion, deletion, and / or substitution at pre-selected sites in the genome of a targeted cell. When a sequence of an endogenous gene is edited, for example by deletion, insertion, or substitution of nucleotide(s) / nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. Discussed herein, a “disrupted gene” refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, “disrupting a gene” refers to a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art, and described, e.g., in United States Patent No.11254912, incorporated herein by reference in its entirety.

[0248] In embodiments, a nuclease-dependent approach can be used to conduct targeted gene editing of a T cell. Such a nuclease-dependent approach can achieve targeted editing through the specific introduction of double strand breaks (DSBs) by specific endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material. Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR / Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Discussed herein, a CRISPR system, or CRISPR nuclease system can include a non-coding RNA molecule (e.g., guide RNA) that binds DNA and Cas proteins (e.g., Cas9) with nuclease functionality (Sander et al., Nature Biotechnology (2014); 32:347-355; Hsu et al., Cell (2014); 157(6):1262-1278).

[0249] In embodiments, a multispecific CAR T cell herein may comprise one or more disrupted genes. For example, one or more genes whose expression is disrupted can comprise adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), cytokine inducible SH2-containing protein (CISH), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS SITE (E.G. AAVS1, AAVS2, ETC.)), or chemokine (C—C motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor 1(LAIR1), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7),Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto-oncogene (SKIL), TGFB induced factor homeobox 1 (TGIF1), interleukin 10 receptor subunit alpha (IL10RA), interleukin 10 receptor subunit beta (IL10RB), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1 (SIT1), forkhead box P3 (FOXP3), PR domain 1 (PRDM1), basic leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), guanylate cyclase 1, soluble, alpha 3 (GUCY1A3), guanylate cyclase 1, soluble, beta 2 (GUCY1B2), guanylate cyclase 1, soluble, beta 3 (GUCY1B3), cytokine inducible SH2-containing protein (CISH), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, Cbl proto-oncogene B (CBL-B), Zinc Finger Protein 91 (ZFP91), Roquin, CD58, ICAM-1, Regnase-1, RASA2, MED12, Fas, Arid1a, or any combination thereof.

[0250] In a preferred embodiment, a gene whose expression is disrupted is CISH, a negative regulator of TCR signaling. Disruption of the CISH gene may provide a functional advantage over control cells that have an intact CISH gene in improving the sensitivity to certain cytokines (e.g., IL-2 / IL-15), increasing T cell proliferation, and / or limiting T cell exhaustion. In some examples, the CISH gene may be disrupted by methods described in Daher M. et al, Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells, Blood. 2021 Feb 4;137(5):624-636, which is incorporated by reference herein in its entirety. In one example, the CISH gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 78-81. In another example, the CISH gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 85-86.

[0251] In embodiments, a gene whose expression is disrupted is CBL-B, a negative regulator of T cell activation. Disruption of the CBL-B gene may provide a functional advantage over control cells that have an intact CBL-B gene in enhancing T cell activation. In some examples, the CBL- B gene may be disrupted by methods described in Augustin R. et al., Targeting Cbl-b in cancer immunotherapy, J Immunother Cancer. 2023 Feb;11(2):e006007; Hooper K. et al., Knockout ofCBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells, Blood (2018) 132 (Supplement 1): 338; and Guo X. et al., CBLB ablation with CRISPR / Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy, J Immunother Cancer. 2021 Mar;9(3):e001975, each of which is incorporated by reference herein in its entirety. In one example, the CBL-B gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequence of any one of SEQ ID NOs: 82-84.

[0252] In embodiments, a gene whose expression is disrupted is Roquin (e.g., Roquin-1). Disruption of the Roquin gene may provide a functional advantage over control cells that have an intact Roquin gene in increasing T cell proliferation and enhancing antitumor activity. In some examples, the Roquin gene may be disrupted by methods described in Mai D et al., Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells, Proc Natl Acad Sci U S A. 2023 Mar 21;120(12):e2218632120, which is incorporated by reference herein in its entirety.

[0253] In embodiments, a gene whose expression is disrupted is ZFP91. Disruption of the ZFP91 gene may provide a functional advantage over control cells that have an intact ZFP91 gene in improving T cell glycolytic fitness and effector function. In some examples, the ZFP91 gene may be disrupted by method described in Wang F. et al., J Clin Invest. 2021 Oct 1;131(19):e144318, which is incorporated by reference herein in its entirety. In embodiments, a gene whose expression is disrupted is CD58. In embodiments, a gene whose expression is disrupted is ICAM-1. In embodiments, both CD58 and ICAM-1 are disrupted. Disruption of the CD58 and / or ICAM-1 genes may provide a functional advantage over control cells that have an intact CD58 and / or ICAM-1 gene in disrupting T cell adhesion and co-stimulatory interactions to reduce Host vs Graft allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Teo HY et al. IL12 / 18 / 21 Preactivation Enhances the Antitumor Efficacy of Expanded γδT Cells and Overcomes Resistance to Anti-PD-L1 Treatment, Cancer Immunol Res.2023 Jul 5;11(7):978-999, which is incorporated by reference in its entirety. In one example, the ICAM-1 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., CRISPR-Cas or CRISPR-Mad7 system) comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 92-93. In one example, the CD58 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., CRISPR-Cas or CRISPR-Mad7 system) comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 94-95.

[0254] In embodiments, a gene whose expression is disrupted is Regnase-1 gene. Regnase-1 (Regulatory RNase 1), also known as ZC3H12A or MCPIP-1, is a ribonuclease that promotes decay of target mRNA through recognition of 3’ UTR stem loop motifs. Disruption of the Regnase- 1 gene may provide a functional advantage over control cells that have an intact Regnase-1 gene in enhancing T cell expansion and persistence. In one example, the Regnase-1 gene may be disrupted as described in Jun Wei et al., Targeting Regnase-1 programs long-lived effector T cells for cancer therapy. Nature.2019 Dec; 576(7787): 471–476, which is incorporated by reference in its entirety.

[0255] In embodiments, a gene whose expression is disrupted is RAS p21 protein activator 2 (RASA2) gene. RASA2 is a RAS GTPase-activating protein (RasGAP) that is a signaling checkpoint in T cells, which can be downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. Disruption of RASA2 may enhance MAPK signaling and CAR T cell cytolytic activity in response to target antigen. RASA2-deficient T cells may exhibit increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent disease-associated cell killing. In some examples, the RASA2 gene may be disrupted by the method described in Julia Carnevale et al., RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature. 2022 Sep;609(7925):174-182, which is incorporated by reference herein in its entirety.

[0256] In embodiments, a gene whose expression is disrupted is MED12 gene. Mediator complex subunit 12 (MED12) is a Mediator subunit, and can regulate metabolic activity and fitness, e.g., increased glycolysis, oxidative phosphorylation, and spare respiratory capacity. Disruption of the MED12 gene may provide a functional advantage over control cells that have an intact MED12 gene in enhancing T cell activation and effector function. In one example, the MED12 gene may be disrupted as described in Katherine A Freitas et al., Enhanced T cell effector activity by targeting the Mediator kinase module, Science. 2022 Nov 11;378(6620):eabn5647, which is incorporated by reference in its entirety.

[0257] In embodiments, a gene whose expression is disrupted is Fas gene. Disruption of the Fas gene may provide a functional advantage over control cells that have an intact Fas gene in reducing T cell allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Jiangtao Ren et al., A versatile system for rapid multiplex genome-edited CAR T cell generation,Oncotarget. 2017 Mar 7; 8(10): 17002–17011, which is incorporated by reference herein in its entirety.

[0258] In embodiments, a gene whose expression is disrupted is Arid1a gene. Arid1a regulates the acquisition of the epigenetic state of terminal exhaustion. Disruption of the Arid1a gene may provide a functional advantage over control cells that have an intact Arid1a gene in improving tumor control and enhancing persistence of T cells. In one example, the Arid1a gene may be disrupted as described in Julia A Belk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell. 2022 Jul 11;40(7):768-786.e7.

[0259] T cells can be obtained from an allogeneic or an autologous donor. The T cells can be, partially or entirely purified, or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in WO 2017 / 197347. The expansion may be performed before or after, or before and after, a CAR construct is introduced into the T cell(s).

[0260] In embodiments, the T cells can be modified to include a nucleic acid construct that encodes a protein which imparts a desired functionality to the T cells of the present disclosure. For example and without limitation, such a nucleic acid may encode for a chimeric DAP10 adaptor polypeptide, described in U.S. Provisional Application No. 63 / 272,613 and U.S. Provisional Application No.63 / 347,194, the contents of each of which is hereby expressly incorporated herein by reference in their entirety. In embodiments, a chimeric DAP10 adaptor polypeptide is capable of associating with a chimeric antigen receptor of the present disclosure, for example a CAR of the present disclosure may comprise a DAP10-interacting domain. In additional or alternative examples, a chimeric DAP10 adaptor polypeptide may not associate with a CAR of the present disclosure, but instead may interact with an endogenous or exogenous polypeptide that includes a DAP10-interacting domain.

[0261] T cells described herein can be stored, e.g., cryopreserved, for use in adoptive cell transfer. D. Methods of Treatment

[0262] The present disclosure also provides methods of treating a disease, e.g., cancer, autoimmune disease, or a pathogen infection. In general, the methods comprise administering aneffective amount of multispecific CAR T cells or a pharmaceutical composition comprising the T cells, to a subject in need thereof. Broadly speaking, the cells targeted by the multispecific CAR T cells of the present disclosure include any kind of cell that comprises a CD70 antigen including, e.g. alloreactive T cells, as well as any kind of cell expressing another disease-associated antigen of interest including, e.g. autoimmune disease-associated cells, cancer cells, pathogens or cells infected by pathogens. The cells targeted by the multispecific CAR T cells may be the same or different cells. In embodiments, the autoimmune disease-associated cells targeted by the at least one affinity binding domain in the second CAR are B cells, e.g. pro B cells, pre B cells, transitional / immature B cells, naive B cells, GC B cells, memory B cells, plasmablasts, and plasma cells.

[0263] In embodiments, the methods are for treating a cancer. Examples of cancers include include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS- related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer,oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0264] The multispecific T cells can be used to target cancer cells. In specific embodiments said cancer cells are hematological malignant cells. In specific embodiments, said cancer cells are solid malignant cells. In certain aspects, said cancer cells are lymphoma cells, renal cell carcinoma cells, or gliobastoma cells, for example. In some aspects the cancer cells are HTLV-1 -associated malignant cells or EBV-associated malignant cells, for example. In specific embodiments, the cancer cells are CD70-positive. In specific embodiments, the cancer being treated is renal cell cancer, thymic carcinoma, nasopharyngeal carcinoma, brain tumor, Hodgkin and non-Hodgkin lymphomas, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia, T-cell leukemia, multiple myeloma, EBV- and HTLV-I associated malignancies, kidney, pancreatic, larynx, pharynx, melanoma, ovarian, lung (including lung adenocarcinoma), colon, breast, or brain.

[0265] In embodiments, the T cell comprising the chimeric receptor targets any cell that comprises a CD70 antigen and / or another disease-associated antigen, whether or not that targeted cell is cancerous. For example, in embodiments CD70 and / or another disease-associated antigen is expressed on cells that are related to autoimmune disorders, as in certain aspects there is dysregulation of CD70-CD27 co-stimulation that contributes to autoimmunity. The dual CAR designs of the subject invention synergistically target both activated T lymphocytes via CD70, including autoreactive T cells commonly implicated in autoimmune diseases, as well as memory B cells and plasmablasts via anti-CD19 and the like

[0266] In embodiments, the methods are for treating an autoimmune disease. Examples of autoimmune diseases include rheumatoid arthritis, rheumatic fever, multiple sclerosis,experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson’s, Alzheimer’s, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott- Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto’s thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenia purpura, dermatomyositis, Sydenham’ a chorea, myasthenia gravis, polyglandular syndromes, bullous pemphigoid, Henoch-Schonlein purpura, poststreptococcalnephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu’s arteritis, Addison’s disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture’s syndrome, thromboangitisubiterans, Sjogren’s syndrome, primary biliary cirrhosis, Hashimoto’s thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pamphigus vulgaris, Wegener’s granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, polymyalgia, peraiciousanemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, Systemic sclerosis, idiopathic inflammatory myopathies (IIM), vasculitis (ANCA+ vasculitis), pemphigus vulgaris, moderate to severe rheumatoid arthritis, neuromyelitis optica spectrum disorder (NMOSD). In embodiments, the autoimmune disease is a B cell associated autoimmune disease. Examples of B cell associated autoimmune diseases include lupus, dermatomyositis, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, Sjogren's syndrome, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pamphigus vulgaris, amyotrophic lateral sclerosis, systemic sclerosis, idiopathic inflammatory myopathies (IIM), membranous nephropathy, pemphigus vulgaris.

[0267] In embodiments, the autoimmune disease is lupus nephritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (e.g., moderate to severerheumatoid arthritis), myasthenia gravis, vasculitis (ANCA+ vasculitis), Sjogren’s syndrome, membranous nephropathy, neuromyelitis optica spectrum disorder (NMOSD), pemphigus vulgaris, systemic sclerosis, and idiopathic inflammatory myopathies (IIM). In embodiments, the autoimmune disease is lupus, lupus nephritis, systemic sclerosis, idiopathic inflammatory myopathies (IIM), or myasthenia gravis. In one example, the autoimmune disease is lupus.

[0268] A course of treatment and / or the severity of one or more symptoms related to autoimmune disease may be monitored. Any appropriate method can be used to determine whether the autoimmune disease is being treated. For example, immunological techniques (e.g., ELISA) can be performed to determine if the level of autoantibodies present within the subject being treated as described herein is reduced following the administration of the pharmaceutical composition, e.g., T cells expressing the CARs disclosed herein. Remission and relapse of the disease can be monitored by testing for one or more markers of the autoimmune disease.

[0269] In embodiments, the methods of treating autoimmune diseases result in a reduction in the number of B cells in a tissue of the subject having the autoimmune disease, e.g., as compared to the levels in the subject prior to treatment or the levels in a similar subject not treated, or receiving a different treatment. In some examples, such reduction in the number of B cell in a tissue of the subject having the autoimmune disease may be at least at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In some examples, such reduction in the number of B cell in a tissue of the subject having the autoimmune disease may be from 1% to 10%, 5% to 15%, 10% to 20%, 15% to 25%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 99%.

[0270] In embodiments, the methods of treating autoimmune diseases result in a reduction in the level of autoantibodies in the subject having the autoimmune disease, e.g., as compared to the levels in the subject prior to treatment or the levels in a similar subject not treated or receiving a different treatment. In some examples, such reduction in the level of autoantibodies may be at least at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In someexamples, such reduction in the level of autoantibodies may be from 1% to 10%, 5% to 15%, 10% to 20%, 15% to 25%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 99%.

[0271] Also provided herein are T cell populations comprising a plurality of multispecific CAR T cells herein. One or multiple T cell populations, having cytotoxic activity against a cell expressing CD70 and / or another disease-associated antigen, can be administered to a subject in any order or simultaneously. If simultaneously, the one or multiple T cell populations can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, s.c, injections or pills. The T cell populations, can be packed together or separately, in a single package or in a plurality of packages. One or all of the T cell populations can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some cases, a T cell populations can proliferate within a subject's body, in vivo, after administration to a subject. One or more T cell populations can be frozen to provide cells for multiple treatments with the same cell preparation. One or more γδ T cell populations of the disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit may include instructions (e.g., written instructions) on the use of the T cell population, and compositions comprising the same.

[0272] In some cases, a method of treating a disease comprises administering to a subject a therapeutically effective amount of a at least one multispecific CAR T cell population herein, wherein the administration treats the disease or condition. In embodiments the therapeutically effective amount of the multispecific CAR T cell population is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, δ hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, δ days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In embodiments the therapeutically-effective amount of the multispecific CAR T cell population is administered for at least one week. In embodiments the therapeutically-effective amount of the multispecific CAR T cell population is administered for at least two weeks.

[0273] A multispecific CAR T cell population described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering apharmaceutical composition containing the T cell population can vary. For example, the multispecific CAR T cell population can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein. In some examples, the administration of a the multispecific CAR T cell population of the disclosure is an intravenous administration. For example, one or multiple dosages of the cell population can be administered as soon as is practicable after the onset of a disease and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. In embodiments, one or multiple dosages of the γδ T cell population can be administered years after onset of the disease or cancer and before or after other treatments.

[0274] In embodiments, the multispecific CAR T cell population is administered simultaneously or sequentially with one or more methods to elevate common gamma chain cytokine(s). As used herein, “one or more methods to elevate common gamma chain cytokine(s): refers to a method, or combination of methods, that alters the physiological state of a subject, such that at least one common gamma chain cytokine level is elevated in the subject. In embodiments, the method elevates the level of one or more common gamma chain cytokine(s) selected from the group consisting of IL-2, IL-7, and IL-15, preferably wherein the method elevates the level of IL- 15 in the subject. In embodiments, the method comprises lymphodepletion. In embodiments, the method comprises administering one or more common gamma chain cytokine(s) to the subject. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, are administered. In embodiments, the method comprises secreting common gamma chain cytokine(s) from an administered, e.g., T cell. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, are secreted.

[0275] In embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before introducing the T cell(s). In embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the T cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced T cell(s), preferably wherein the method comprisesadministering IL-2 or one or more mimetics thereof, more preferably wherein the method comprises administering IL-15 or one or more mimetics thereof. The amount of administered common gamma chain cytokine(s) can be an amount effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced T cell(s) before and / or after introducing the T cell(s). Exemplary amounts of IL-15 include, without limitation between 0.01 – 10 µg / kg / dose every 24 hours for IL-15. Exemplary amounts of IL-2 include, without limitation, between about 3x106and about 22x106units every 8 - 48 hours. For example, the dosing regimen for IL2 in RCC is 600,000 International Units / kg (0.037 mg / kg) IV q8hr infused over 15 minutes for a maximum 14 doses.

[0276] In embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before administering the T cell(s) and administering simultaneously with introducing the T cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced T cell(s).

[0277] The multispecific CAR T cells of the subject invention can also be advantageously administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities (e.g., for treating cancer) including, e.g., chemotherapy, radiation therapy, or immunotherapy. A patient can also be preconditioned with a therapeutically effective amount of multispecific CAR T cells prior to receiving the chemotherapy, radiation therapy, or immunotherapy (e.g., cell therapy). Suitable immunotherapies for use in combination with multispecific CAR T cells include autologous and allogeneic cell therapies, engineered T and NK cells, immune engagers, fusion proteins, or other immune-oncology agents.

[0278] In embodiments, the multispecific CAR T cells herein can be administered in conjunction with an alternative cellular immunotherapy for treating a disease (e.g. CAR NK cells, or Treg therapy). For example, a method of treatment herein can include a conditioning step, e.g. a preconditioning step, of administering a therapeutically effective amount of T cells to a subject simultaneously or sequentially with administration of a cellular immunotherapy directed against the disease. In embodiments, the cellular immunotherapy can comprise further administering an engineered T cell or NK cell therapy comprising a CAR binding to any disease-associated antigen of interest. In embodiments, the subject engineered T cells may comprise a dual CAR binding toCD70 and another disease-associated antigen, e.g., an immune disease-associated antigen or tumor-associated antigen.

[0279] In embodiments, the multispecific CAR T cells herein may be advantageously administered in conjunction with adoptive cell therapies (ACT) (for reviews of HSCT and adoptive cell therapy approaches, see, Rager & Porter, Ther Adv Hematol (2011) 2(6) 409-428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) 11(4):473-487; Wang et al. Int. J. Cancer: (2015)136, 1751-1768; and Chang, Y. J. and X. J. Huang, Blood Rev, 2013.27(1): 55-62), each of which is incorporated by reference herein in its entirety. Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, e.g., CAR T, CAR NK). Beyond the necessity for donor-derived cells to reconstitute hematopoiesis after radiation and chemotherapy, immunologic reconstitution from transferred cells is important for the elimination of residual tumor cells. The efficacy of ACT as a curative option for malignancies is influenced by a number of factors including the origin, composition and phenotype (lymphocyte subset, activation status) of the donor cells, the underlying disease, the pre-transplant conditioning regimen and post-transplant immune support (e.g., IL-2 therapy) and the graft-versus-tumor (GVT) effect mediated by donor cells within the graft. Additionally, these factors may be balanced against transplant-related mortality, typically arising from the conditioning regimen and / or excessive immune activity of donor cells within the host (e.g., graft-versus-host disease, cytokine release syndrome, etc.). E. Methods of CAR Detection

[0280] Quantification of CARs transgene(s) level in host cells of the present disclosure can be accomplished in a variety of ways. Examples include but are not limited to genomic detection (e.g., quantitative PCR, digital PCR), transcriptomic detection (e.g., RNA sequencing, RNA in situ hybridization), proteomic detection (e.g., fluorescence microscopy), and organismal detection (e.g., positron emission tomography (PET) scanning, two-photon microscopy), and various combinations thereof.

[0281] In embodiments, an isolated nucleic acid encoding one or more CARs may include one or more labels or markers to facilitate an ability to monitor CARs expression level. However, it is herein recognized that inclusion of a label or marker, for example LNGFR, can disadvantageouslyimpact viral titer levels given the increased molecular weight of the resultant CARs, particularly in a case where other polypeptides are also associated with the CARs (e.g., dnTGFβR2). Other additional or alternative methodology, for example reliance on vector copy number (VCN) is also associated with disadvantages, including but not limited to the fact that the ratio of VCN to CARs may require substantial process development time and may change throughout scale up / process refinement.

[0282] In one embodiment, quantification of a CAR may be determined via immunological detection of a linker region (e.g., T2A, P2A, E2A, F2A), for example a cleavage linker region that couples the first CD27 CAR to a second CAR targeting a disease-associated antigen. An advantage of such an approach is that the linker region is directly coupled to the CAR, and can enable intracellular C-terminal detection of said CAR. For example and without limitation, an antibody that can be used to detect 2A peptides includes anti-2A (3H4) (Novus Biologicals, Littleton, CO).

[0283] In the case of 2A peptides, the self-cleaving mechanism is the result of ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide. Because of efficient cleavage, in preferred embodiments a P2A peptide is used. The efficient cleavage of P2A peptides may reduce or eliminate exogenous epitopes associated with a gene of interest, which in turn may reduce immunogenicity and hence impart a lesser likelihood that a patient treated with a CAR-engineered host cell will exhibit an immune reaction against the engineered host cell (see WO 2020 / 223478).

[0284] When a dual CAR construct encodes a polypeptide comprising a first CAR comprising CD27 and a second CAR targeting another antigen, the quantification of the first CAR may be determined using a tag sequence (e.g., to distinguish the CD27 domain in the first CAR from endogenous CD27 in a host cell) coupled with the first CAR. In embodiments, the polypeptide encoded by the dual CAR construct comprises, from C-terminus to N-terminus, the first CAR, a self-cleavage sequence, the second CAR. In such cases, the first CAR may be detected using an antibody recognizing the skip sequence of the self-cleavage sequence. For example, the self- cleavage sequence may be P2A and the first CAR may be detected using an antibody recognizing the P2A skip sequence, e.g., the 2F13 rabbit monoclonal antibody (e.g., Sigma-Aldrich Cat. # ZRB1746). In embodiments, the polypeptide encoded by the dual CAR construct comprises, from C-terminus to N-terminus, the second CAR, a self-cleavage sequence, the first CAR, and further comprises a tag at the N-terminal side of the first CAR. In such cases, the first CAR may bedetected using an antibody recognizing the tag at the N-terminal side of the first CAR. In some examples, the tag may be a modified T2A sequence recognized by antibody 5E6 (e.g., Sigma- Aldrich Cat. # MABE1923). F. Dosages

[0285] One or multiple multispecific CAR T cell populations of the present disclosure, may be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms comprise additional lymphocytes. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose re-closable containers can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not comprise a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

[0286] One or multiple multispecific CAR T cell populations of the present disclosure, may be present in a composition in an amount of at least 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1 x 103cells, at least 2 x 103cells, at least 3 x 103cells, at least 4 x 103cells, at least 5 x 103cells, at least 6 x 103cells, at least 7 x 103cells, at least 8 x 103cells, at least 9 x 103cells, at least 1 x 104cells, at least 2 x 104cells, at least 3 x 104cells, at least 4 x 104cells, at least 5 x 104cells, at least 6 x 104cells, at least 7 x 104cells, at least 8 x 104cells, at least 9 x 104cells, at least 1 x 105cells, at least 2 x 105cells, at least 3 x 105cells, at least 4 x 105cells, at least 5 x 105cells, at least 6 x 105cells, at least 7 x 105cells, at least 8 x 105cells, at least 9 x 105cells, at least 1 x 106cells, at least 2 x 106cells, at least 3 x 106cells, at least 4 x 106cells, at least 5 x 106cells, at least 6 x 106cells, at least 7 x 106cells, at least 8 x 106cells, at least 9 x 106cells, at least 1 x 107cells, at least 2 x 107cells, at least 3 x 107cells, at least 4 x 107cells, at least 5 x 107cells, at least 6 x 107cells, at least 7 x 107cells, at least 8 x 107cells, at least 9 x 107cells, at least 1 x 108cells, at least 2 x 108cells, at least 3 x 108cells, at least 4 x 108cells, at least 5 x 108cells, atleast 6 x 108cells, at least 7 x 108cells, at least 8 x 108cells, at least 9 x 108cells, at least 1 x 109cells, or more.

[0287] The therapeutically effective dose of one or multiple multispecificCAR T cell populations herein can be from about 1 cell to about 10 cells, from about 1 cell to about 100 cells, from about 1 cell to about 10 cells, from about 1 cell to about 20 cells, from about 1 cell to about 30 cells, from about 1 cell to about 40 cells, from about 1 cell to about 50 cells, from about 1 cell to about 60 cells, from about 1 cell about 70 cells, from about 1 cell to about 80 cells, from about 1 cell to about 90 cells, from about 1 cell to about 100 cells, from about 1 cell to about 1 x 103cells, from about 1 cell to about 2 x 103cells, from about 1 cell to about 3 x 103cells, from about 1 cell to about 4 x 103cells, from about 1 cell to about 5 x 103cells, from about 1 cell to about 6 x 103cells, from about 1 cell to about 7 x 103cells, from about 1 cell to about 8 x 103cells, from about 1 cell to about 9 x 103cells, from about 1 cell to about 1 x 104cells, from about 1 cell to about 2 x 104cells, from about 1 cell to about 3 x 104cells, from about 1 cell to about 4 x 104cells, from about 1 cell to about 5 x 104cells, from about 1 cell to about 6 x 104cells, from about 1 cell to about 7 x 104cells, from about 1 cell to about 8 x 104cells, from about 1 cell to about 9 x 104cells, from about 1 cell to about 1 x 105cells, from about 1 cell to about 2 x 105cells, from about 1 cell to about 3 x 105cells, from about 1 cell to about 4 x 105cells, from about 1 cell to about 5 x 105cells, from about 1 cell to about 6 x 105cells, from about 1 cell to about 7 x 105cells, from about 1 cell to about 8 x 105cells, from about 1 cell to about 9 x 105cells, from about 1 cell to about 1 x 106cells, from about 1 cell to about 2 x 106cells, from about 1 cell to about 3 x 106cells, from about 1 cell to about 4 x 106cells, from about 1 cell to about 5 x 106cells, from about 1 cell to about 6 x 106cells, from about 1 cell to about 7 x 106cells, from about 1 cell to about 8 x 106cells, from about 1 cell to about 9 x 106cells, from about 1 cell to about 1 x 107cells, from about 1 cell to about 2 x 107cells, from about 1 cell to about 3 x 107cells, from about 1 cell to about 4 x 107cells, from about 1 cell to about 5 x 107cells, from about 1 cell to about 6 x 107cells, from about 1 cell to about 7 x 107cells, from about 1 cell to about 8 x 107cells, from about 1 cell to about 9 x 107cells, from about 1 cell to about 1 x 108cells, from about 1 cell to about 2 x 108cells, from about 1 cell to about 3 x 108cells, from about 1 cell to about 4 x 108cells, from about 1 cell to about 5 x 108cells, from about 1 cell to about 6 x 108cells, from about 1 cell to about 7 x 108cells, from about 1 cell to about 8 x 108cells, from about 1 cell to about 9 x 108cells, or from about 1 cell to about 1 x 109cells.

[0288] In some cases, the therapeutically effective dose of therapeutically effective dose of one or multiple multispecific CAR T cell populations herein can be from about 1 x 103cells to about 2 x 103cells, from about 1 x 103cells to about 3 x 103cells, from about 1 x 103cells to about 4 x 103cells, from about 1 x 103cells to about 5 x 103cells, from about 1 x 103cells to about 6 x 103cells, from about 1 x 103cells to about 7 x 103cells, from about 1 x 103cells to about 8 x 103cells, from about 1 x 10 cells to about 9 x 103cells, from about 1 x 10 cells to about 1 x 104cells, from about 1 x 103cells to about 2 x 104cells, from about 1 x 103cells to about 3 x 104cells, from about 1 x 103cells to about 4 x 104cells, from about 1 x 103cells to about 5 x 104cells, from about 1 x 103cells to about 6 x 104cells, from about 1 x 103cells to about 7 x 104cells, from about 1 x 103cells to about 8 x 104cells, from about 1 x 103cells to about 9 x 104cells, from about 1 x 103cells to about 1 x 105cells, from about 1 x 103cells to about 2 x 105cells, from about 1 x 103cells to about 3 x 105cells, from about 1 x 103cells to about 4 x 105cells, from about 1 x 103cells to about 5 x 105cells, from about 1 x 103cells to about 6 x 105cells, from about 1 x 103cells to about 7 x 105cells, from about 1 x 103cells to about 8 x 105cells, from about 1 x 103cells to about 9 x 105cells, from about 1 x 103cells to about 1 x 106cells, from about 1 x 103cells to about 2 x 106cells, from about 1 x 103cells to about 3 x 106cells, from about 1 x 103cells to about 4 x 106cells, from about 1 x 103cells to about 5 x 106cells, from about 1 x 103cells to about 6 x 106cells, from about 1 x 103cells to about 7 x 106cells, from about 1 x 103cells to about 8 x 106cells, from about 1 x 103cells to about 9 x 106cells, from about 1 x 103cells to about 1 x 107cells, from about 1 x 103cells to about 2 x 107cells, from about 1 x 103cells to about 3 x 107cells, from about 1 x 103cells to about 4 x 107cells, from about 1 x 103cells to about 5 x 107cells, from about 1 x 103cells to about 6 x 107cells, from about 1 x 103cells to about 7 x 107cells, from about 1 x 103cells to about 8 x 107cells, from about 1 x 103cells to about 9 x 107cells, from about 1 x 103cells to about 1 x 108cells, from about 1 x 103cells to about 2 x 108cells, from about 1 x 103cells to about 3 x 108cells, from about 1 x 103cells to about 4 x 108cells, from about 1 x 103cells to about 5 x 108cells, from about 1 x 10 cells to about 6 x 108cells, from about 1 x 10 cells to about 7 x 108cells, from about 1 x 103cells to about 8 x 108cells, from about 1 x 103cells to about 9 x 108cells, or from about 1 x 103cells to about 1 x 109cells.

[0289] In some cases, the therapeutically effective dose of one or multiple multispecific CART cell populations herein can be from about 1 x 106cells to about 2 x 106cells, from about 1 x106cells to about 3 x 106cells, from about 1 x 106cells to about 4 x 106cells, from about 1 x 106cells to about 5 x 106cells, from about 1 x 106cells to about 6 x 106cells, from about 1 x 106cells to about 7 x 106cells, from about 1 x 106cells to about 8 x 106cells, from about 1 x 106cells to about 9 x 106cells, from about 1 x 106cells to about 1 x 107cells, from about 1 x 106cells to about 2 x 107cells, from about 1 x 106cells to about 3 x 107cells, from about 1 x 106cells to about 4 x 107cells, from about 1 x 106cells to about 5 x 107cells, from about 1 x 106cells to about 6 x 107cells, from about 1 x 106cells to about 7 x 107cells, from about 1 x 106cells to about 8 x 107cells, from about 1 x 106cells to about 9 x 107cells, from about 1 x 106cells to about 1 x 108cells, from about 1 x 106cells to about 2 x 108cells, from about 1 x 106cells to about 3 x 108cells, from about 1 x 106cells to about 4 x 108cells, from about 1 x 106cells to about 5 x 108cells, from about 1 x 106cells to about 6 x 108cells, from about 1 x 106cells to about 7 x 108cells, from about 1 x 106cells to about 8 x 108cells, from about 1 x 106cells to about 9 x 108cells, from about 1 x 106cells to about 1 x 109cells, from about 1 x 106cells to about 2 x 109cells, from about 1 x 106cells to about 3 x 109cells, from about 1 x 106cells to about 4 x 109cells, from about 1 x 106cells to about 5 x 109cells, from about 1 x 106cells to about 6 x 109cells, from about 1 x 106cells to about 7 x 109cells, from about 1 x 106cells to about 8 x 109cells, from about 1 x 106cells to about 9 x 109cells, from about 1 x 107cells to about 1 x 109cells, from about 1 x 107cells to about 2 x 109cells, from about 1 x 107cells to about 3 x 109cells, from about 1 x 107cells to about 4 x 109cells, from about 1 x 107cells to about 5 x 109cells, from about 1 x 107cells to about 6 x 109cells, from about 1 x 107cells to about 7 x 109cells, from about 1 x 107cells to about 8 x 109cells, from about 1 x 107cells to about 9 x 109cells, from about 1 x 108cells to about 1 x 109cells, from about 1 x 108cells to about 2 x 109cells, from about 1 x 108cells to about 3 x 109cells, from about 1 x 108cells to about 4 x 109cells, from about 1 x 108cells to about 5 x 109cells, from about 1 x 108cells to about 6 x 109cells, from about 1 x 108cells to about 7 x 109cells, from about 1 x 108cells to about 8 x 109cells, from about 1 x 108cells to about 9 x 109cells, or from about 1 x 108cells to about 1 x 1010cells. G. Preservation

[0290] In embodiments, the one or multiple multispecific CAR T cell populations herein may be formulated in freezing media and placed in cryogenic storage units such as liquid nitrogen freezers (-195C) or ultra-low temperature freezers (-65C, -80C or -120C) for long-term storage ofat least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freeze media can contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate and / or hydroyethyl starch (HES) with physiological pH buffering agents to maintain pH between about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0 or about 6.5 to about 7.5. In embodiments, the cryopreserved cells can be thawed and further processed, for example by stimulation with antibodies, proteins, peptides, and / or cytokines as mentioned herein. The cryopreserved cells can be thawed and genetically modified with viral vectors (including retroviral and lentiviral vectors) or non-viral means (including RNA, DNA, and proteins) as described herein. Alternatively, host cells as described herein can be, e.g., optionally expanded by the methods described herein, genetically modified, and then cryopreserved.

[0291] Thus, the multispecific CAR T cells as disclosed herein can be cryopreserved to generate cell banks in quantities of at least about 1, 5, 10, 100, 150, 200, 500 vials at about at least 101, 102, 103, 104, 105, 106, 107, 108, 109, or at least about 1010cells per mL in freeze media. The cryopreserved cell banks may retain their functionality and can be thawed and, optionally, be activated / stimulated and / or expanded. In some aspects, thawed cells can be stimulated and expanded in suitable closed vessels such as cell culture bags and / or bioreactors to generate quantities of cells as allogeneic cell product. In other examples, the cryopreserved cells comprise an autologous cell product. Cryopreserved cells can maintain their biological functions for at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months, or at least about 60 months under cryogenic storage condition. In some aspects, no preservatives are used in the formulation. In embodiments, the cryopreserved cells can be thawed and infused into multiple patients as allogeneic off-the-shelf cell product. EXAMPLES

[0292] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Example 1. In vitro activity of Vδ1 CD27 receptor CAR T cells

[0293] This Example demonstrates that Vδ1 CD27 CAR T cells exhibit robust activity against cells expressing CD70 in vitro.

[0294] As shown at FIG. 1, target cell lines Raji, HeLa, ACHN, A498, and 786-O (ATCC) were stained with anti-CD70 antibody (Biolegend) or isotype (R&D Systems). Cells were analyzed on the Novocyte (Agilent). CD70 expression in different cell lines was graphed as MFI (mean fluorescence intensity) ratio. The resulting ratio of antibody MFI to isotype MFI was graphed. CD70 low-expressing: HeLa and ACHN, CD70 medium-expressing: Raji, and CD70 high- expressing: A498 and 786-O.

[0295] For the long-term cytotoxicity assay, Vδ1 CD27 receptor CAR T cells were cocultured with target cell lines (HeLa, FIG.2A; Raji, FIG.2B; A498, FIG.2C) at an effector to target ratio (E:T) of 2:1 for 120 hours. Target cells were modified to express Nuc-NIR by transducing them with Incucyte® Nuclight NIR lentivirus (Sartorius). Viable Nuc-NIR expressing target cells were quantified every 4 hours by the Incucyte® SX5 (Sartorius). Cytotoxicity index was calculated by dividing the total NIR object area of each time point by the value of time 0 hours. Vδ1 CD27 receptor CAR T cells showed strong cytotoxic activity against different cancer cell lines such as HeLa (cervical), Raji (Lymphoma), and A498 (Renal cell). The lower cytotoxicity index indicates better cytotoxic potential of Vδ1 CD27 receptor CAR T cells (boxes) versus target cells alone (circles).

[0296] It was assessed as to whether soluble CD27 could impact cytotoxic potential of Vδ1 CD27 receptor CAR T cells. Briefly, Vδ1 CD27 receptor CAR T cells were cocultured with Nuc- NIR expressing A498 target cell line at an E:T ratio of 1:2. At time 0 hours, soluble CD27 (Acro Biosystems) was added to the coculture at different concentrations ranging from 0-8 µg / mL. Viable Nuc-NIR expressing target cells were quantified every 2 hours by the Incucyte® SX5 (Sartorius). Cytotoxicity index at time points 18-hour and 36-hour was calculated by dividing the total NIR object area of each time point by the value at time 0 hours. The lack of difference in the cytotoxic index between co-cultures at 0µg and 0.25-8µg / mL of soluble CD27 indicates that the presence of soluble CD27 does not interfere with the cytotoxic potential of Vδ1 CD27 receptor CAR T cells (FIG.3).Example 2. In vivo control of renal cell carcinoma by Vδ1 CD27 receptor CAR T cells

[0297] This Example demonstrates that full-length CD27 CAR with 4-1BB costimulatory domain and CD3ζ signaling domain exhibits improved in vivo tumor control as compared to truncated CD27 CAR with CD8 hinge / TM domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

[0298] The in vivo efficacy of Vδ1 CD27 receptor CAR T cells was assessed in a subcutaneous renal cancer model using A498 cells. The results are shown in FIG. 4. Female NSG mice (The Jackson Laboratory) were implanted subcutaneously with 4 x 106A498 cells (ATCC), mixed with Matrigel (1:1 ratio) (Corning). Animals were randomized into groups of 5 when tumor volume reached an average of 100 mm3and were injected IV (tail vein) with 2.5 x 106, 5 x 106, or 10 x 106Vδ1 CD27 receptor CAR T cells. Negative control groups were treated with non-functioning Vδ1 CD27 truncated receptor CAR T cells at a dose of 5 x 106CAR+ cells (Group E) and a tumor alone group (Group A, no treatment). Human IL-2 (Proleukin) was administered intraperitoneally prior to treatment and then thrice a week. Tumor volume was measured twice a week with calipers. Tumor response was durable at all three dose levels of Vδ1 CD27 receptor CAR T cells (Groups B-D) as compared to control groups (tumor alone and the group treated with non-functioning Vδ1 CD27 truncated receptor CAR T cells). These data support the cytotoxic potential of Vδ1 CD27 receptor CAR T cells in a human renal cancer cell xenograft model.

[0299] The in vivo efficacy of Vδ1 CD27 receptor CAR T cells was also assessed in a subcutaneous renal cancer model using ACHN cells. The results are shown in FIG. 5. Female NSG mice (The Jackson Laboratory) were implanted subcutaneously with 5 x 106ACHN cells (ATCC), mixed with Matrigel (1:1 ratio) (Corning). Animals were randomized into groups of 5 when tumor volume reached an average of 120 mm3and were injected IV (tail vein) with 2.5 x 106, 5 x 106, or 10 x 106Vδ1 CD27 receptor CAR-T cells. Negative control groups were treated with Vδ1 CD27 truncated receptor CAR-T cells at a dose of 5 x 106CAR+ cells (Group E) or left untreated (Group A). Human IL-2 was administered intraperitoneally prior to treatment and then thrice a week. Tumor volume was measured twice a week with calipers. Tumor response was durable at the higher dose levels of Vδ1 CD27 receptor CAR T cells (Group C, Group D) as compared to control groups (tumor alone and the group treated with non-functioning Vδ1 CD27 truncated receptor CAR-T cells). These data support the cytotoxic potential of Vδ1 CD27 receptor CAR-T cells in a human renal cancer cell xenograft model that has low CD70 expression.Example 3. Identification of CD27 CAR expression

[0300] This Example demonstrates that antibodies to a 2A peptide included as part of a CD27 CAR can be used to determine CD27 CAR surface expression.

[0301] Vδ1 T cells modified with three different CD20 CAR constructs, two of which contain P2A and one that does not, were thawed and counted by Annexin-Dapi on the Novocyte (Agilent). Approximately 5 x105cells were stained with cell viability dye Zombie Aqua (Biolegend). The cells were surface stained for Vδ1 (Biolegend) and anti-CAR idiotype antibody (Adicet Bio) and fixed with Fixation Buffer (Biolegend). The cells were then permeabilized with Permeabilization Buffer (BD Biosciences). After permeabilization, the cells were stained with anti-P2A antibody (Novus Biosciences) and acquired on the Novocyte (Agilent). The P2A positive population co- stained with CAR surface staining for samples CD20 CAR-P2A-DAP10.7 and CD20 CAR-P2A- DAP10.9 (FIG.6). The results suggest intracellular staining of P2A can be used for CAR detection. Example 4. Gene editing of γδ T cells

[0302] This Example demonstrates the use of a CRISPR / Cas9 system to generate a CISH knockout in γδ T cells.

[0303] To knockout the CISH gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex comprised of CRISPR / Cas9 (Integrated DNA Technologies) and CISH sgRNA’s (Synthego Corporation) were combined with activated Vδ1 T cells from PBMC’s (P3 Primary Cell 4D- Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells by electroporation (Lonza 4D-Nucleofector). Post electroporation, the cells were transferred to T flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery and expansion in a 37oC CO2incubator (ThermoFisher). To evaluate CISH KO efficiency, cell pellets were collected following αβ T cell depletion (StemCell Technologies) on day 7 post gene edit and genomic DNA was extracted using the NucleoSpin Tissue kit (MACHEREY-NAGEL). The edited region of the CISH gene was amplified by PCR and Sanger sequenced (Sequetech). Gene knockout efficiency was determined using the ICE Analysis tool (Synthego). High efficiency knockout of the CISH gene was observed from Vδ1 T cells (FIG.7).

[0304] Post CISH gene knockout, Vδ1 T cell enrichment in PBMCs was monitored over time by flow cytometry (anti-Vδ1, BioLegend). CISH gene knockout did not affect Vδ1 T cell enrichment as compared to unedited Mock control cells. Similar purity of CISH gene edited Vδ1T cells was achieved post TCRαβ depletion (StemCell Technologies) as compared to Mock control cells (FIG.8A).

[0305] Cell viability of Vδ1 T cell enriched PBMCs was monitored post CISH gene knockout (Countess II, ThermoFisher). CISH KO cells maintained high cell viability similarly to unedited Mock control cells (FIG.8B). Example 5. Identification of CAR expression

[0306] ADI-270 cells (cells expressing PL1179, SEQ ID NO: 54) were thawed and counted by Annexin-Dapi on the Novocyte (Agilent). Approximately 2 x105cells were stained with cell viability dye Zombie Aqua (Biolegend). The cells were fixed with Fixation Buffer (Biolegend). The cells were then permeabilized with Permeabilization Buffer (BD Biosciences). After permeabilization, the cells were stained with anti-P2A antibody (Novus Biosciences) and acquired on the Novocyte. The P2A positive population represent the transduction efficiency of the CAR. The results are shown in FIG.9, which suggest that intracellular staining of P2A can be used for CAR detection of ADI-270. Example 6. In vitro activity of ADI-270

[0307] Target cell lines Caki-1, Caki-2, 769-P, ACHN, A498, 786-O, H1792, H1975, H460, and HuT78 (ATCC) were stained with anti-CD70 antibody conjugated to PE (Biolegend) or isotype control conjugated to PE (R&D Systems). Cells were analyzed on the Novocyte. The number of CD70 molecules per cell was quantified using the flow cytometric BD Quantibrite™ Bead Kit (BD Biosciences) (FIG. 10). Several tumor cell lines that expressed a wide range of CD70 levels were identified for subsequent in vitro functional evaluation for ADI-270.

[0308] For the long-term cytotoxicity assay, ADI-270 was co-cultured with target cell lines at an effector to target ratio (E:T) of 2:1, 1:1, and 1:2 for 120 hours. Target cells were modified to express Nuc-NIR by transducing them with Incucyte® Nuclight NIR lentivirus (Sartorius). Viable Nuc-NIR expressing target cells were quantified every 4 hours by the Incucyte® SX5 (Sartorius). Cytotoxicity index was calculated by dividing the total NIR object area of each time point by the value of time 0 hours. Increased tumor cell killing is represented by a lower Cytotoxicity Index. As shown in FIG.11, ADI-270 showed potent cytotoxic activity when co-cultured against multiple RCC cell lines (A498, 769-P, Caki-2) expressing a range of CD70 levels, and against CD70- positive NSCLC (H1945) and T cell lymphoma (HuT78) cell lines. No cytotoxicity against theCD70 knock-out A498 cell line was observed at the E:T ratios tested, suggesting the cytolytic activity of ADI-270 is specific and dependent on the expression of CD70.

[0309] ADI-270 is equipped with dnTGFβRII armoring to mitigate the immunosuppressive effects of TGFβ1 that can exist within the tumor microenvironment (TME). To demonstrate the functionality of the dnTGFβRII expressed by ADI-270, cytolytic activity against CD70-positive cell lines was compared to anti-CD70 CAR Vδ1 γδ T cells (no dnTGFβRII) (PL1107, SEQ ID NO: 50) in the absence and presence of exogenously added TGFβ1 (R&D Systems) using an in vitro repetitive stimulation assay. A498- and ACHN-Nuc NIR fluorescently labeled RCC tumor cell lines and effector cells were added into a 96-well plate at an E:T ratio of 5:1. TGFβ1 was added to the respective co-culture conditions at a final concentration of 20ng / mL. The first stimulation was monitored for 2 days after which fresh target cells were plated in another 96-well plate. The effector cells from the first stimulation were then transferred into the plate with fresh target cells. The second stimulation was monitored for an additional 2 days. The co-cultures were monitored every 4 hours using Incucyte® SX5. The Cytotoxicity Index was calculated based on the remaining viable target tumor cells by dividing the total NIR object area of each time point by the value at time 0 for each stimulation. The results are shown in FIG. 12. In the presence of TGFβ1, ADI-270 maintained cytolytic activity against A498 and ACHN when compared to the no TGFβ1 condition after the second stimulation with tumor cells. The anti-CD70 CAR Vδ1 γδ T cells in the presence of TGFβ1 were associated with the lowest tumor control after the second stimulation due to the lack of armoring to mitigate the inhibitory effect by TGFβ1. The data suggests ADI-270 maintains its potent in vitro cytotoxicity against RCC cell lines in the presence of TGFβ1, demonstrating the functionality of dnTGFβRII armoring in Vδ1 γδ T cells.

[0310] TGFβ1 is known to directly inhibit T cell proliferation. To further assess the functionality of dnTGFβR2 armoring, in vitro proliferation of ADI-270 was evaluated in the absence and presence of exogenously added TGFβ1 (R&D Systems). ADI-270 and anti-CD70 CAR Vδ1 γδ T cells (effector cells) were labeled with Cell Trace Violet (CTV) (ThermoFisher), to measure their proliferative capacity. The CTV-labeled effector cells were cocultured with A498 target cells at an E:T ratio of 1:2 for 7 days. The coculture was harvested, and the effector cells were evaluated for proliferation using flow cytometry (Novocyte) by determining the % of cell divisions with decreased CTV fluorescent intensity using FlowJo™ Proliferation Modeling software (FlowJo). The results are shown in FIG. 13. In the presence of TGFβ1, ADI-270maintained in vitro proliferation after stimulation with A498 tumor cells when compared to without TGFβ1 added based on the similar % of cell divisions detected. In contrast, the anti-CD70 CAR Vδ1 γδ T cells had reduced % cell divisions detected when stimulated with A498 cells in the presence of TGFβ1 compared to the no TGFβ1 condition. The results indicate that dnTGFβRII armoring reduces the effects of TGFβ1 to inhibit proliferation of Vδ1 γδ T cells. The data is a representative of 4 different donors. The numbers represent the % of cells within each gated cell division.

[0311] The binding of TGFβ1 to the heteromeric complex of TGFβ type II, and type I receptors induce a signaling cascade that phosphorylates SMAD2 and SMAD3 (pSMAD2 / 3). To investigate if dnTGFβRII armoring in ADI-270 can reduce the phosphorylation of SMAD2 / 3, ADI-270 and anti-CD70 CAR Vδ1 γδ T cells were serum starved for 2 hours and cultured in the presence and absence of exogenous TGFβ1 (R&D Systems) for 15 minutes. The cells were then fixed, permeabilized, and stained for anti-pSMAD2 / 3 and analyzed using flow cytometry (Novocyte). The results are shown in FIG.14. After exposure to TGFβ1, ADI-270 was associated with lower levels of pSMAD2 / 3 compared to anti-CD70 CAR Vδ1 γδ T cells which further supports the functionality of the dnTGFβRII. The modest increase in pSMAD2 / 3 with ADI-270 compared to the no TGFβ1 condition is likely attributed to the presence of untransduced CAR Vδ1 γδ T cells within the total population that are not armored with dnTGFβR2. Data shown is the average Geo Mean from 3 different donors. Statistical analysis was performed using paired t-test ** p<0.005.

[0312] To determine if dnTGFβRII armoring of ADI-270 can minimize changes in transcripts levels associated with T cell effector functions, ADI-270 and anti-CD70 CAR Vδ1 γδ T cells were activated with rhuCD70 (AcroBio) for 24 hrs in the presence and absence of TGF-β1 (R&D Systems). RNA was extracted from the cells using the RNeasy Plus Kits (Qiagen). The nCounter® CAR-T Characterization Panel (Nanostring) and nCounter® SPRINT Profiler (Nanostring) were used to quantitate T cell-related transcripts from the samples. The nCounter® Advanced Analysis Software (Nanostring) was used to identify differentially expressed genes (DEGs). The results are shown in FIG. 15. Volcano plots represent DEGs from ADI-270 vs anti-CD70 CAR Vδ1 γδ T cells (not armored) activated with rhuCD70 (5µg / mL) in the presence or absence of TGF-β1 (20ng / mL) for 24 hrs from 2 different donors. Blue (up pointing) triangles represent DEGs upregulated in TGF-β1 treated vs untreated conditions. Red (down pointing) triangles represent DEGs downregulated in TGF-β1 treated vs untreated conditions. Stimulated anti-CD70 CAR Vδ1γδ T cells were associated with increased transcript changes when compared between + / - TGF-β1 conditions. Minimal transcript changes were observed from stimulated ADI-270 between + / - TGF- β1 conditions further supporting dnTGFβRII armoring is minimizing TGF-β1-induced transcript changes in Vδ1 γδ T cells.

[0313] ADI-270 has multiple effector functions, such as the production of soluble mediators, and recognition and killing of CD70+ tumor cells, that contributes to effective anti-tumor activity. To investigate the cytokine and chemokine production of ADI-270 and anti-CD70 CAR Vδ1 γδ T cells, a coculture with A498 and ACHN target cell lines at an E:T ratio of 1:1 was performed, and supernatants were collected after 24 hours post-stimulation. Supernatants were analyzed using the Milliplex Immunology Multiplex Assay Panel on the Luminex® FLEXMAP 3D instrument. Analysis of the supernatants revealed that IFN-γ, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, and IL-10 were measured at similar levels for both ADI-270 and anti-CD70 CAR Vδ1 γδ T cells after stimulation with A498 and ACHN cell lines. The results are shown in FIGS.16A-16B. The data suggests that the dnTGFβRII armoring in ADI-270 does not alter the cytokine and chemokine release profile when compared to anti-CD70 CAR Vδ1 γδ T cells. Minimal cytokine and chemokine secretion was seen with ADI-270 and target alone. Data shown is the mean concentration (pg / mL) of cytokine and chemokines derived from two different donors. Example 7. In vivo control of human tumor xenografts in NSG mice with ADI-270 treatment

[0314] ADI-270 was designed with a dnTGFβRII armor against the immunosuppressive factor TGF-β1. Research grade ADI-270 and anti-CD70 CAR Vδ1 γδ T cells (no dnTGFβRII) were manufactured from three separate donors. The activity of ADI-270 with or without armoring was then tested in a CD70-expressing human renal cell carcinoma (RCC) cell line A498 implanted subcutaneously in female NSG mice (Charles River) (n=5 per group). Mice were randomized into study cohorts when tumor volumes were approximately 150 mm3.5E6 ADI-270, 5E6 anti-CD70 CAR Vδ1 γδ T cells, and 5E6 untransduced γδ1 T cells were administered on Day 0 as a single bolus dose. Human IL-2 supplementation was given three times a week for the duration of the study. Tumor volume and mouse weight were monitored over the duration of the experiment. The results are shown in FIGS.17A-17B. (FIG.17A) Mean tumor volumes (MTV) with standard error bars for each group are shown over the duration of the study until the endpoint was reached on Day 40, when mean tumor volumes in the tumor alone cohort reached approximately 2000 mm3.(FIG.17B) Statistical analysis of Day 40 tumor volume data (end-of-study) was performed using one-way ANOVA, with Tukey’s multiple comparison test. ADI-270 caused no adverse health effects and showed significant tumor control compared to the untreated controls or mice treated with untransduced γδ T cells. Furthermore, ADI-270 demonstrated functional advantages with significant enhancement of efficacy when compared to unarmored anti-CD70 CAR Vδ1 γδ T cells.

[0315] Technical grade ADI-270 material was evaluated in A498 human RCC subcutaneous xenograft tumor model at three dose levels (5e6,10e6 and 15e6 CAR+ viable cells). Human renal cell carcinoma A498 subcutaneous xenografts were established by inoculation of 4E6 cells / mouse on the right hind flank of female NSG mice. Mice were randomized into study cohorts when tumor volumes were approximately 150 mm3(N=5 mice per group). ADI-270 was administered on Day 0 as a single bolus dose. Human IL-2 supplementation was given three times a week for the duration of the study. Tumor volume and mouse weight were monitored over the duration of the experiment. The results are shown in FIGS. 18A-18B. (FIG. 18A) Average tumor volumes for the duration of the study and (FIG.18B) statistical comparison (Tukey’s multiple comparison test) between treatment groups and the vehicle control group at the end of the study (Day 39) when mean tumor volumes in the tumor alone cohort reached approximately 2000 mm3was performed. **** represents a p value of ≤0.0001. Technical grade ADI-270 caused no adverse health effects and showed similar potency as research grade material previously tested in this model. Higher dose levels of ADI-270 (10e6 and 15e6) led to complete eradication of tumors in this RCC solid tumor model.

[0316] The activity of ADI-270 was assessed against a different indication with lower levels of CD70 expression. HuT78, a CD70-low expressing human T cell lymphoma cell line, was implanted subcutaneously by inoculation of 2E6 HuT78 cells on the right hind flank of female NSG mice. Mice were randomized into study cohorts when tumor volumes reached approximately 200mm3(N=5 per group). ADI-270 was administered on Day 0 as a single bolus dose. Human IL- 2 supplementation was given to appropriate groups three times a week for the duration of the study. The results are shown in FIGS.19A-19B. (FIG.19A) Mean tumor volumes (MTV) with standard error bars for each group are shown over the duration of the study until the endpoint was reached on Day 21, when mean tumor volumes in the tumor alone cohort reached approximately 2000mm3. (FIG.19B) Statistical analysis of Day 21 tumor volume data (end-of-study) was performed using one-way ANOVA with Dunnett’s multiple comparison test. **** represents a p value of ≤0.0001.ADI-270 showed significant control with and without IL-2 supplementation in a dose-dependent fashion. Example 8. Ex vivo tissue analysis from ADI-270 treated NSG mice implanted with human tumor xenografts

[0317] Based on the robust in vivo tumor control without incidence of xenogeneic GvHD in A498 tumor-bearing NSG mice, the proliferation and activation of ADI-270 specifically within the tumor, with little to no accumulation in normal tissues was confirmed. Tissues from A498- bearing NSG mice receiving a single IV dose of Cell Trace Violet (CTV) (ThermoFisher) labeled ADI-270 were harvested at day 7 and 14. The results are shown in FIGS.20A-20C. (FIG.20A) CTV labeled ADI-270 shows increased cell proliferation in tumor as indicated by the dilution of the CTV dye (histogram plots) compared to day 0 control. (FIG.20B) Harvested ADI-270 were stained for activation-associated markers and (FIG.20C) ADI-270 was seen to specifically home to the tumor when compared to lung, spleen, blood, and bone marrow at day 7 and 14. Example 9. Manufacturability and anti-tumor activity assessment between CD27 receptor and anti-CD70 scFv CAR approaches

[0318] The design of the anti-CD70 CAR binding domain used to modify T cells can affect manufacturability due to the induction of fratricide against CD70+ activated T cells in the culture. To evaluate the impact of the CD27 receptor and anti-CD70 scFv CAR approach on γδ T cell expansion, healthy donor PBMCs were used to activate, and engineer Vδ1 T cells to express the CD27 receptor CAR or anti-CD70 scFv CARs (CAR1 and CAR2, VHVL and VLVH scFv orientation respectively) derived from the scFv described in EP2100619A1. The results are shown in FIGS. 21A-21B. Cell culture samples were harvested at different timepoints during the expansion process and analyzed using flow cytometry (Novocyte) to quantitate (FIG. 21A) absolute counts and (FIG. 21B) viability (DAPI viability dye) of the Vδ1 T cells. The results showed Vδ1 T cells expressing the CD27 receptor were associated with increased cell counts during the expansion compared to the anti-CD70 scFv CAR Vδ1 T cells. In addition, reduced viability was observed with the anti-CD70 scFv CAR Vδ1 T cells, suggesting a potential induction of fratricide compared to Vδ1 T cells expressing the CD27 receptor CAR which showed higher viability throughout the expansion process.

[0319] To assess the anti-tumor activity between CD27 receptor and anti-CD70 scFv CAR approach, healthy donor PBMCs were used to activate, and engineer Vδ1 or αβ T cells to expressthe CD27 receptor CAR (ADI-270) or anti-CD70 scFv CARs respectively (αβ CAR-T Ref 1 and Ref2) derived from the antibodies described in WO2021095011A1 and US20190233528. ADI- 270 and αβ CAR-T Refs were cocultured with target RCC tumor cell lines expressing high and low levels of surface CD70 antigen, A498 and ACHN respectively at an E:T ratio of 1:1 for 24 hrs. The target tumor cells were modified to express Nuc-NIR by transducing them with Incucyte® NucLight NIR lentivirus (Sartorius). Viable Nuc-NIR expressing target cells were quantified every 4 hours by the Incucyte® SX5 (Sartorius). The cytotoxicity index (CI) was calculated by dividing the total NIR object area of each time point by the value of time 0 hours. The % cytotoxicity was calculated by [(CI of target alone at time x)- (CI of sample at time x). x 100] / (CI of target alone at time x). The results are shown in FIG.22. The data points represent the average % cytotoxicity (y-axis) of the effector cells from 3 donors at each time point post-stimulation (x-axis) with the specific target cell line indicated. Both ADI-270 and the αβ CAR-T Refs showed potent cytotoxic activity against the A498 (CD70 high expressing). In contrast, against ACHN (CD70 low expressing), ADI-270 was associated with increased anti-tumor activity compared to the αβ CAR- T Refs. The results suggest a functional advantage of the CD27 receptor CAR compared to the anti-CD70 scFv-based approach in targeting tumors expressing a lower range of CD70 antigen. Example 10: In vivo control of human hematologic-derived tumor xenografts in NSG mice with ADI-270 treatment

[0320] The anti-tumor activity of ADI-270 was assessed against hematologic-derived tumor cells with lower levels of CD70 expression. The results are shown in FIGS.23A-23C. (FIG.23A) Target cell lines MM1.S (multiple myeloma), THP-1 (AML), and A498 (RCC) (ATCC) were stained with anti-CD70 antibody conjugated to PE (Biolegend) or isotype control conjugated to PE (R&D Systems). Cells were analyzed using flow cytometry (Novocyte). The number of CD70 molecules per cell was quantified using the flow cytometric BD Quantibrite™ Bead Kit (BD Biosciences). (FIG. 23B) MM1.S or (FIG. 23C) THP-1 tumor cells were implanted subcutaneously by inoculation of 1E6 or 2E6 cells respectively on the right hind flank of female NSG mice. Mice were randomized into study cohorts when tumor volumes reached approximately 200mm3(N=5 per group). ADI-270 (5E6 and 10E6 CAR+ cells) was administered on Day 0 as a single bolus dose. Human IL-2 supplementation was given to appropriate groups three times a week for the duration of the study. Mean tumor volumes (MTV) with standard error bars for each group are shown over the duration of the study until the endpoint was reached on Day 28, whenmean tumor volumes in the tumor alone cohort reached approximately 2000mm3. Statistical analysis of Day 28 tumor volume data (end-of-study) was performed using one-way ANOVA with Dunnett’s multiple comparison test. **** represents a p value of ≤0.0001. The results demonstrate the robust in vivo anti-tumor activity associated with ADI-270 to significantly inhibit tumor growth against CD70 low-expressing tumor cells. Example 11. Host vs Graft (HvG) armoring of ADI-270

[0321] ADI-270 and irrelevant CAR (targeting CD20) Vδ1 γδ T cells (target cells) were co- cultured with T cells (effectors) isolated from autologous and allogeneic (Donor A and B) PBMC donors using the EasySep™ Human T Cell Enrichment Kit (StemCell Technologies) at a fixed ratio E:T ratio of 5:1 and 10:1 with IL-2 supplementation. Half of the culture media (10% FBS RPMI-1640) was replaced every 2-3 days with fresh IL-2. On day 5, the cells were harvested and analyzed by flow cytometry (Novocyte) to quantitate the absolute counts of ADI-270 and irrelevant CAR Vδ1 γδ T cells from the co-culture with autologous and allogeneic T cells. The % HvG susceptibility was determined by comparing the absolute counts of ADI-270 and irrelevant CAR Vδ1 γδ T cells effectors cells of the T cell autologous co-cultures (control for minimal cell killing) to the T cell allogeneic co-cultures. The results are shown in FIG. 24. Reduced HvG susceptibility of ADI-270 was observed compared to Vδ1 T cells expressing an irrelevant CAR suggesting ADI-270 potentially targets CD70+ activated alloreactive host T cells and can limit HvG Vδ1 T cell rejection to aid in persistence. Statistical comparison between ADI-270 and irrelevant CAR groups was determined using 2-way ANOVA.

[0322] The CD27 receptor CAR expressed by ADI-270 has the potential to target and kill activated CD70-expressing alloreactive host T cells for improved persistence. Primed allogeneic T cells were generated by coculturing enriched T cells (EasySep™ Human T Cell Enrichment Kit, StemCell Technologies) from 3 PMBC donors with allogeneic macrophage-derived monocytes (Isolated CD14+ monocytes using human CD14 Microbeads (Miltenyi Biotech) and cultured in RPMI 164010% FBS media with 50 ng / mL recombinant human M-CSF (R&D Systems) for 6 days) at a fixed T cell-macrophage ratio of 10:1 with IL-2 (10 ng / mL) supplementation for 7 days. Half of the culture media was replaced every 2-3 days with fresh IL-2. ADI-270 and irrelevant CAR Vδ1 γδ T cells (target cells-derived from the same macrophage-derived monocyte PBMC donor) were labeled with Cell Trace Violet and cocultured with the primed effector T cells (fromallogeneic donors) at fixed E:T ratios of 1:1, 1:2, and 1:4. After 24 hours, the cells were harvested and analyzed by flow cytometry (Novocyte) to quantitate the absolute counts of ADI-270 and irrelevant CAR Vδ1 T cells from the coculture. The % cell survival was determined by comparing absolute counts of ADI-270 and irrelevant CAR Vδ1 T cells coculture to the absolute counts of the respective target alone control. The results are shown in FIG.25. Results showed the irrelevant CAR Vδ1 T cells had lower cell survival in the presence of primed allogeneic T cells than ADI- 270 suggesting that ADI-270 can reduce allogeneic T cell rejection to improve persistence. Statistical comparison between ADI-270 and irrelevant CAR groups was determined using 2-way ANOVA, **p<0.01, ****p<0.0001.

[0323] The results suggest that using a dual CAR binding to CD70 and another tumor- associated antigen, or co-administering a CAR binding to CD70 and a CAR binding to another tumor associated antigen may have reduced alloreactivity compared to using the CAR binding to another tumor-associated antigen alone. Example 12

[0324] This example shows the study of dual CAR construct (PL1605) encoding an anti-CD70 CAR and an anti-CD19 CAR, and δ1 γδ T cells expressing the dual CAR construct. The vector titer of PL1605 is shown in Table 5 below. The anti-CD19 CAR of PL1605 expressed similarly to the anti-CD70 CAR.

[0325] Table 5 Total TU PL No Plasmid Name Staining / HT1080 Titers (TU / mL) * pSINBA- Anti- G4S CD27 P2A flCD27-41BB- FMC63 Linker PL1605 CD3z-P2A- 34e+06 Kymriah- 6.59E+06 5.43E+06 4.97E+06 4.53E+06 CTL019.CO

[0326] Cell expansion with the dual CAR construct was tested. FIG.27A shows the yield of δ1 γδ T cells expressing the dual CAR construct compared head-to-head with ADI-001 (δ1 γδ T cell expressing an anti-CD20 CAR as described in Nishimoto KP et al., Allogeneic CD20-targetedγδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models, Clin Transl Immunology.2022 Feb 2;11(2):e1373) (~70%). FIG.27B shows the growth of δ1 γδ T cells expressing the dual CAR construct in extended cultures.

[0327] After transduction of δ1 γδ T cells with the dual CAR construct, proportional expression of both anti-CD70 and anti-CD19 CARs were detected. The dual CAR construct expressed in the δ1 γδ T cells at a level similar to a single anti-CD70 CAR or single anti-CD19 CAR control (FIG.28A). The expression of the dual CAR construct generally increased in cells derived from two donors when more MOI was used in transduction (FIG.28B).

[0328] Cytotoxicity of δ1 γδ T cells expressing the dual CAR construct was tested using tumor cell lines. The target antigen expression profiles of the tumor cell lines are shown in Table 6 below. Target-specific killing of δ1 γδ T cells expressing the dual CAR construct was detected in the tumor cell lines. The dual CAR construct functioned similarly to anti-CD19 CAR against cell lines that express CD19. The results are shown in FIG.29. The data shown is averaged across 2 donors.

[0329] Table 6 Cell line CD19 CD70 Raji parental ++ + Raji CD70 KO ++ - MM.1S parental - + MM.1S-FcRL5 - + A498 - +++

[0330] Cytotoxicity of δ1 γδ T cells expressing the dual CAR construct was tested with B cells. FIGS.30A and 30B show the dual CAR construct was functionally equivalent to anti-CD19 CAR against healthy B cells and additionally targeted activated primary T cells. FIG.31 shows that the dual CAR construct was equivalent to anti-CD19 CAR against B cells from patient with multiple sclerosis). Example 13: Enhancement of CAR Vδ1 T cells effector function using membrane bound IL-12 (mbIL-12)

[0331] CAR vector, consisting of the anti-CD19 (clone FMC63) scFv (as described in Kochendefer JN et al., Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells, Blood.2010Nov 11;116(19):3875-86), CD8α hinge and transmembrane domains, and 4-1BB and CD3ζ signaling domains followed by a self-cleaving P2A sequence that is used to separate the mbIL-12 molecule (as described in Lee et al., Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting, bioRxiv.2023 Jan 7;2023.01.06.522784), was constructed by cloning a DNA fragment containing all CAR domains (Integrated DNA Technologies) into a self-inactivating (SIN) Moloney Murine Leukemia Virus (MMLV) gamma-retroviral plasmid. The constructs design is shown in FIG. 32 (construct A) CAR (FMC63) construct, (construct B) CAR-mbIL-12 construct, and (construct C) mbIL-12 construct.

[0332] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand and engineer Vδ1 γδ T cells to express the CD19 CAR-mbIL-12 transgene. The results are shown in FIGS. 33A-33D. (FIG. 33A) Vδ1 γδ T cells (post-transduction) were sampled at different time points over the course of the expansion to determine cell counts. The total Vδ1 γδ T cell counts did not differ between CAR and CAR-mbIL-12, suggesting mbIL-12 has no negative impact on the expansion of Vδ1 γδ T cells. Cells were harvested, stained for CAR and mbIL-12 using anti-FMC63 antibody conjugated to PE (Acrobio Systems, Newark, DE) and anti-IL12 conjugated to APC (Miltenyi Biotec) respectively, and acquired on the Novocyte flow cytometer (Agilent Technologies) to determine the percentage of CAR and mbIL-12 positive Vδ1 γδ T cells. (FIG.33B) No difference in % CAR was observed between Vδ1 γδ T cells expressing CAR and CAR-mbIL-12 at the end of the process to further support mbIL-12 did not affect CAR surface expression. (FIG. 33C) Over the course of the expansion, the % mbIL-12 expressed on the cell surface of Vδ1 γδ T cells decreased over time. (FIG.33D) CAR-mbIL-12 Vδ1 γδ T cells were co- cultured with and without CD19+ Raji cells for 18 hrs. After stimulation with target cells, mbIL- 12 increased on the cell surface of the Vδ1 γδ T cells compared to unstimulated conditions suggesting the surface expression of mbIL-12 is dependent on CAR activation.

[0333] To demonstrate the functionality of CAR-mbIL-12, cytolytic activity against CD19- positive cell lines using CAR, CAR-mbIL-12, and mbIL-12 transduced Vδ1 γδ T cells was evaluated in an in vitro repetitive stimulation assay. Raji-Nuc NIR fluorescently labeled cell line and effector cells were added into a 96-well plate at E:T ratios of 5:1, 2.5:1, and 1.25:1. The first stimulation was monitored for 3 days after which fresh target cells were plated in another 96-well plate. The effector cells from the first stimulation were then transferred into the plate with fresh target cells. The second stimulation was monitored for an additional 3 days, and the sameprocedure was performed for a third stimulation. The co-cultures were monitored every 4 hours using Incucyte® SX5 (Satorius). The Cytotoxicity Index was calculated based on the remaining viable target tumor cells by dividing the total NIR object area of each time point by the value at time 0 for each stimulation. The lower the cytotoxicity index, the better the cytotoxic potential. After the second and third stimulation, the CAR-mbIL-12 Vδ1 γδ T cells were associated with greater tumor cell killing compared to CAR and mbIL12 Vδ1 γδ T cells across the E:T ratios tested. Co-culture conditions with a Cytotoxicity Index > 1 (indication of incomplete tumor control), were not analyzed in the subsequent stimulation. The results are shown in FIG.34 and demonstrate the functionality of the CAR-mbIL-12 expressed by Vδ1 γδ T cells to sustain cytolytic killing of tumor cells.

[0334] CAR-mbIL-12 Vδ1 γδ T cells were evaluated in Raji cell subcutaneous xenograft tumor model at a suboptimal dose of 2.5e6 CAR+ viable cells. Raji cell subcutaneous xenografts were established by inoculation of 1E6 cells / mouse on the right hind flank of female NSG mice. Mice were randomized into study cohorts when tumor volumes were approximately 200 mm3(N=5 mice per group). Vδ1 γδ T cells expressing CAR and CAR-mbIL-12 were administered on Day 0 as a single bolus dose. Human IL-2 supplementation was given three times a week for the duration of the study. Tumor volume and mouse weight were monitored over the duration of the experiment. The results are shown in FIG. 35. The CAR-mbIL-12 Vδ1 γδ T cells were associated with a substantially greater inhibition of tumor cell growth compared to the CAR Vδ1 γδ T cell group at the suboptimal treatment dose. The data supports the enhanced functionality of Vδ1 γδ T cells expressing CAR-mbIL-12. Example 14: CISH KO enhances the function of Vδ1 T cells

[0335] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand, CRISPR-gene edit, and engineer Vδ1 γδ T cells to express a CAR. To knockout the CISH gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex comprising CRISPR-Cas9 (Integrated DNA Technologies) or -MAD7 (Aldevron) and CISH sgRNA were combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells by electroporation (Lonza 4D-Nucleofector). Post electroporation, the cells were transferred to T flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery prior to transduction with gammaretroviral vector encoding the CAR and then were subsequently expanded. To evaluate CISH KO efficiency, cell pellets were collected, and genomic DNA wasextracted using the NucleoSpin Tissue kit (MACHEREY-NAGEL). The edited region of the CISH gene was amplified by PCR and Sanger sequenced (Sequetech). Gene knockout efficiency was determined using the ICE Analysis tool (Synthego). To evaluate the KO of CISH protein in CAR Vδ1 T cells, WT (unedited) and CISH KO CAR Vδ1 T cells were incubated with and without IL- 2 (Peprotech) for 5 hours. Cell lysates were generated for western blot detection of CISH protein using anti-CISH Rabbit mAb (Cell Signaling). An IRDye 800CW goat anti-Rabbit IgG secondary antibody (LI-COR) was used for detection of the primary antibody and the presence of the IRDye was imaged with the Odyssey imaging system (LI-COR). The results are shown in FIGS. 26A- 26B. (FIG. 36A) WT CAR Vδ1 T cells showed an increase in levels of CISH protein when exposed to IL-2. In contrast, CISH KO CAR Vδ1 T cells had substantially reduced levels of CISH protein in the presence of IL-2 supporting the KO of CISH. (FIG.36B) To demonstrate enhanced function of CISH KO CAR-transduced and untransduced Vδ1 T cells, the cytolytic activity against the B7-H6+ HCT-15 cell line (ATCC) was evaluated using an in vitro repetitive stimulation assay. HCT-15 NIR fluorescently labeled cell line and effector cells were added into a 96-well plate at an E:T ratio of 5:1 with or without different concentrations of IL-2. Each stimulation was monitored for 3 days after which fresh target cells were plated in another 96-well plate. The effector cells from each stimulation were then transferred into the plate with fresh target cells. The co-cultures were monitored every 4 hours using Incucyte® SX5 (Satorius). The Cytotoxicity Index was calculated based on the remaining viable target tumor cells by dividing the total NIR object area of each time point by the value at time 0 for each stimulation. The lower the cytotoxicity index, the better the cytotoxic potential. After the second stimulation, CISH KO untransduced Vδ1 T cells were associated with greater tumor cell killing compared to WT untransduced Vδ1 T cells only in the presence of IL-2. Without IL-2, CISH KO CAR Vδ1 T cells had superior tumor control compared to WT CAR Vδ1 T cells. The results suggest CISH KO enhances the in vitro cytotoxicity of Vδ1 T cells.

[0336] CAS9 CISH sgRNA: TGTACAGCAGTGGCTGGTGG (AGG) (SEQ ID NO: 85)

[0337] MAD7 CISH sgRNA: (TTTA) GGTGTACAGCAGTGGCTGGTG (SEQ ID NO: 86). Example 15: CBL-B KO enhances the function of Vδ1 T cells

[0338] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand, CRISPR-gene edit, and engineer Vδ1 γδ T cells to express a CAR. To knockout the CBL- B gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex comprising CRISPR-Cas9 (IntegratedDNA Technologies) and CBL-B sgRNAs were combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells by electroporation (Lonza 4D-Nucleofector). Post electroporation, the cells were transferred to T flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery prior to transduction with gammaretroviral vector encoding the CAR and then were subsequently expanded. The results are shown in FIGS.37A-37B. (FIG. 37A) To determine CBL-B KO efficiency, genomic DNA was extracted from cell pellets using the NucleoSpin Tissue kit (MACHARY-NAGEL). The edited region was amplified by PCR using previously in-house optimized primers. The PCR reaction was run on an agarose gel, and the prominent band excised, and gel extracted using NucleoSpin Gel and PCR Cleanup kit (MACHARY-NAGEL). The extracted DNA was sequenced by Sanger sequencing (Sequetech). % Indels were determined using the ICE Analysis tool (Synthego). (FIG. 37B) To demonstrate enhanced function of CBL-B KO CAR-transduced and untransduced Vδ1 T cells, the cytolytic activity against the PSMA+ PC3 cell line was evaluated using an in vitro repetitive stimulation assay. PC3-PSMA NIR fluorescently labeled cell line and effector cells were added into a 96-well plate at an E:T ratio of ~3:1 with or without IL-2. Each stimulation was monitored for 3 days after which fresh target cells were plated in another 96-well plate. The effector cells from each stimulation were then transferred into the plate with fresh target cells. The co-cultures were monitored every 4 hours using Incucyte® SX5 (Satorius). The Cytotoxicity Index was calculated based on the remaining viable target tumor cells by dividing the total NIR object area of each time point by the value at time 0 for each stimulation.

[0339] Cas9 Multi-guide sgRNA sequences:

[0340] AAGACUCUUUAAAGAAGGCA (SEQ ID NO: 82)

[0341] AGUACUCAUUCUCACUGAGU (SEQ ID NO: 83)

[0342] CGUAAAUGCUGAUAUGUAUC (SEQ ID NO: 84)

[0343] Cas9 Single-guide: TAATCTGGTGGACCTCATGA (AGG) (SEQ ID NO: 87). Example 16: Roquin-1 KO enhances the function of Vδ1 T cells

[0344] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand, CRISPR-gene edit, and engineer Vδ1 γδ T cells to express a CAR. To knockout the Roquin-1 gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex comprising CRISPR-Cas9 (Integrated DNA Technologies) and Roquin-1 sgRNAs were combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was deliveredinto Vδ1 T cells by electroporation (Lonza 4D-Nucleofector). Post electroporation, the cells were transferred to T flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery prior to transduction with gammaretroviral vector encoding the CAR and then were subsequently expanded. The results are shown in FIGS. 38A-38B. (FIG. 38A) To determine the Roquin KO efficiency genomic DNA was extracted from cell pellets using the NucleoSpin Tissue kit (MACHARY-NAGEL). The edited region was amplified by PCR using previously in-house optimized primers. The PCR reaction was run on an agarose gel, and the prominent band was excised, and gel extracted using NucleoSpin Gel and PCR Cleanup kit (MACHARY-NAGEL). The extracted DNA was sequenced by Sanger sequencing (Sequetech). % Indels were determined using the ICE Analysis tool (Synthego). Edits were first generated using a 1:1:1 ratio of 3 gRNA sequences to demonstrate the ability to edit the gene in Vd1 T cells, prior to single-guide optimization to identify a gRNA with the highest KO efficiency. (FIG. 38B) To demonstrate enhanced function of Roquin-1 KO CAR-transduced and untransduced Vδ1 T cells, the cytolytic activity against the PSMA+ PC3 cell line was evaluated using an in vitro repetitive stimulation assay. PC3-PSMA NIR fluorescently labeled cell line and effector cells were added into a 96-well plate at an E:T ratio of ~3:1 with or without IL-2. Each stimulation was monitored for 3 days after which fresh target cells were plated in another 96-well plate. The effector cells from each stimulation were then transferred into the plate with fresh target cells. The co-cultures were monitored every 4 hours using Incucyte® SX5 (Satorius). The Cytotoxicity Index was calculated based on the remaining viable target tumor cells by dividing the total NIR object area of each time point by the value at time 0 for each stimulation.

[0345] Multi-guide sgRNA sequences:

[0346] AAGCCCAUCAGUUUGGGUUG (SEQ ID NO: 88)

[0347] UGUACAAGCUCCACAAUGGA (SEQ ID NO: 89)

[0348] CAAAUGGGCAAGCCUUGCGG (SEQ ID NO: 90)

[0349] In-house optimized single gRNA sequence: CCTGAATAAACTCCACCGCA (AGG) (SEQ ID NO: 91) Example 17: ICAM-1 and CD58 KO enhances the in vitro cell survival of CAR Vδ1 T cells in the presence of allogeneic PBMCs

[0350] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand, CRISPR-gene edit, and engineer Vδ1 γδ T cells to express a CAR. To knockout the CISHgene in Vδ1 T cells, a ribonucleoprotein (RNP) complex comprised of CRISPR-Cas9 (Integrated DNA Technologies) or -MAD7 (Aldevron) and ICAM-1 or CD58 sgRNA were combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells by electroporation (Lonza 4D-Nucleofector). Post electroporation, the cells were transferred to T flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery prior to transduction with gammaretroviral vector encoding the CAR and then were subsequently expanded. To determine the KO efficiency of ICAM-1 and CD58, WT (unedited), ICAM-1 KO, and CD58 KO CAR Vδ1 γδ T cells were stained with anti-ICAM-1, anti-CD58, or isotype control antibody conjugated to PE (BioLegend) and acquired using the Novocyte flow cytometer (Agilent Technologies). The results are shown in FIGS.39A-39B. (FIG.39A) Greater than 80% KO efficiency was observed for both ICAM-1 and CD58 KO in CAR Vδ1 γδ T cells. (FIG.39B)

[0351] To evaluate if the of ICAM-1 and CD58 can enhance the survival of CAR Vδ1 T cells in the presence of allogeneic PBMCs, a 10-day mixed-lymphocyte reaction assay (MLR) was performed. WT (unedited), CISH KO, ICAM-1 KO, and CD58 KO CAR Vδ1 γδ T cells (target cells) were co-cultured with allogeneic PBMCs (effector cells) at an E:T ratio of 10:1 in the presence of IL-2. During the co-culture, half of the medium was replaced with fresh media containing IL-2 every 2-3 days. At days 3, 6, and 10, cells were harvested from the co-culture and stained for CAR+ and Vδ1+ γδ T cells using B7-H6 conjugated to APC (Adicet Bio generated) for CAR detection and anti-Vδ1 antibody conjugated to PE-CY7 (Adicet Bio generated) for Vδ1 T cell detection. The cells were acquired using the Novocyte flow cytometer to obtain absolute counts of double positive CAR+ Vδ1+ T cells. To determine the % target cell change at each time point, the absolute count of WT CAR+ Vδ1+ γδ T cells was compared to the KO CAR+ Vδ1+ γδ T cell count conditions using the formula: [(WT CAR+ Vδ1+ γδ T cells count – KO CAR+ Vδ1+ γδ T cell count) ÷ (WT CAR+ Vδ1+ γδ T cells count)] x 100. Based on the % target cell change relative to the WT CAR+ Vδ1+ γδ T cells counts, only the ICAM-1 KO and CD58 KO CAR+ Vδ1+ γδ T cells had an increased % target change, suggesting the disruption of ICAM-1 and CD58 reduced the in vitro killing by allogeneic PBMCs for improved cell survival.

[0352] CAS9 ICAM-1 sgRNA: AAAGUCAUCCUGCCCCGGGG (SEQ ID NO: 92)

[0353] MAD7 ICAM-1 sgRNA: TTTG / AATAGCACATTGGTTGGCTAT (SEQ ID NO: 93)

[0354] CAS9 CD58 sgRNA: AAAUAUAUGGUGUUGUGUAU (SEQ ID NO: 94)

[0355] MAD7 CD58 sgRNA: TTTA / GACACTGTGTCAGGTAGCCTC (SEQ ID NO: 95)

[0356] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims. Table 7: Sequence information (Amino acid / Nucleotide Sequence) SEQ Name Sequence ID No. 1 Full-length human MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRF CD70 (NCBI AQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPA Accession LGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASR NP_001243.1) HHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGD (GI:4507605) TLCTNLTGTLLPSRNTDETFFGVQWVRP 2 Signal Peptide MARPHPWWLCVLGTLVGLS (amino acid) 3 Signal Peptide ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCT 4 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE co-stimulatory L domain (amino acid) 5 4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA co-stimulatory ACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGA domain TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG (nucleotide) GATGTGAACTG 6 Furin P2A RAKRSGSGATNFSLLKQAGDVEENPGP (amino acid) 7 Furin P2A CGCGCGAAGCGATCAGGCAGCGGGGCGACAAATTTCAG (nucleotide) CCTTCTGAAACAAGCAGGCGACGTGGAAGAAAACCCCG GTCCA 8 P2A cleavage motif GSGATNFSLLKQAGDVEENPGP - 1 (amino acid) 9 P2A cleavage motif GGCAGCGGGGCGACAAATTTCAGCCTTCTGAAACAAGCA (nucleotide) GGCGACGTGGAAGAAAACCCCGGTCCA 10 CD3ζ signaling RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR domain -1 GRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK (amino acid) GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD3ζ signaling AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA domain -1 CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT (nucleotide) AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CD3ζ signaling RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR domain -2 (1XX) GRDPEMGGKPQRRKNPQEGLFNELQKDKMAEAFSEIGMK (amino acid) GERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR CD3ζ signaling RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR domain -3 GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG (amino acid) ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY (amino acid) GKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQ HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK EGFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT (nucleotide) GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCA CAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCA CCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCC TGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACC ACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCG AAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG ACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATC GACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCT GGAGTACAACTACAACAGCCACAACGTCTATATCATGGC CGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGA TCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCG ACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCG TGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCG CCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGTAA GMCSFR alpha MLLLVTSLLLCELPHPAFLLIP chain signal sequence (amino acid)GMCSFR alpha atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatccca chain signal sequence (nucleotide) Truncated EGFR RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAF (EGFRt) RGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH (amino acid) AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDV IISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATG QVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAH YIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPN CTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGI GLFM Truncated EGFR cgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctccataaatgctacgaa (EGFRt) tattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcctgccggtggcattta (nucleotide) ggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgta aaggaaatcacagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcct ttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagttttctcttgcagtcgt cagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgtgata atttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctc cggtcagaaaaccaaaattataagcaacagaggtgaaaacagctgcaaggccacaggccag gtctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactgcgtctc ttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgag ccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggc catgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattga cggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtc tggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatg cactgggccaggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactg ggatggtgggggccctcctcttgctgctggtggtggccctggggatcggcctcttcatgTGA TAA dnTGFBR2 signal MGRGLLRGLWPLHIVLWTRIAS peptide (amino acid) dnTGFBR2 signal atgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgtggacgcgtatcgc peptide cagc (nucleotide) dnTGFBR2 TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD (amino acid) NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHD PKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCY RVNRQQKLSS dnTGFBR2 acgatcccaccgcacgttcagaagtcggttaataacgacatgatagtcactgacaacaacggt (nucleotide) gcagtcaagtttccacaactgtgtaaattttgtgatgtgagattttccacctgtgacaaccagaaa tcctgcatgagcaactgcagcatcacctccatctgtgagaagccacaggaagtctgtgtggct gtatggagaaagaatgacgagaacataacactagagacagtttgccatgaccccaagctccc ctaccatgactttattctggaagatgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtgagactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatcatcttctcaga agaatataacaccagcaatcctgacttgttgctagtcatatttcaagtgacaggcatcagcctcct gccaccactgggagttgccatatctgtcatcatcatcttctactgctaccgcgttaaccggcagc agaagctgagttcatagtaa LTBR signal MLLPWATSAPGLAWGPLVLGLFGLLAASQP peptide (amino acid) LTBR signal ATGCTGCTGCCTTGGGCTACATCTGCTCCAGGACTTGCTT peptide GGGGACCTCTGGTGCTGGGACTGTTTGGACTGCTGGCTG (nucleotide) CCTCTCAGCCT Full-length LTBR QAVPPYASENQTCRDQEKEYYEPQHRICCSRCPPGTYVSAK (amino acid) CSRIRDTVCATCAENSYNEHWNYLTICQLCRPCDPVMGLEE IAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTE AELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCEN QGLVEAAPGTAQSDTTCKNPLEPLPPEMSGTMLMLAVLLP LAFFLLLATVFSCIWKSHPSLCRKLGSLLKRRPQGEGPNPV AGSWEPPKAHPYFPDLVQPLLPISGDVSPVSTGLPAAPVLE AGVPQQQSPLDLTREPQLEPGEQSQVAHGTNGIHVTGGSM TITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGPPG LSTPHQEDGKAWHLAETEHCGATPSNRGPRNQFITHD Full-length LTBR CAAGCCGTTCCTCCATACGCCAGCGAGAACCAGACCTGC (nucleotide) CGGGACCAAGAGAAAGAGTACTACGAGCCCCAGCACCG GATCTGCTGTAGCAGATGTCCTCCTGGCACATACGTGTC CGCCAAGTGCAGCAGAATCCGGGATACCGTGTGTGCCAC CTGTGCCGAGAACAGCTACAACGAGCACTGGAACTACCT GACCATCTGCCAGCTGTGCAGGCCCTGCGATCCTGTGAT GGGACTCGAGGAAATCGCCCCTTGCACCAGCAAGCGCA AGACCCAGTGCAGATGCCAGCCAGGCATGTTCTGTGCCG CCTGGGCTCTCGAATGTACCCACTGTGAACTGCTGAGCG ACTGCCCTCCTGGAACAGAGGCCGAGCTGAAGGATGAA GTCGGCAAGGGCAACAACCACTGCGTGCCATGTAAAGCC GGCCACTTCCAGAACACAAGCAGCCCTAGTGCCAGGTGC CAGCCTCACACCAGATGCGAGAATCAGGGCCTCGTGGAA GCTGCCCCTGGAACTGCTCAGAGCGACACCACATGCAAG AACCCTCTGGAACCCCTGCCTCCTGAGATGAGCGGCACA ATGCTGATGCTGGCCGTTCTGCTGCCTCTGGCCTTCTTTC TGCTGCTGGCCACCGTGTTCAGCTGCATCTGGAAGTCTC ACCCCAGCCTGTGCAGAAAGCTGGGCAGCCTGCTTAAGA GAAGGCCTCAAGGCGAGGGCCCTAATCCTGTGGCTGGAT CTTGGGAGCCTCCTAAGGCTCACCCTTACTTCCCCGATCT GGTGCAGCCTCTGCTGCCAATCAGCGGAGATGTGTCCCC TGTGTCTACCGGACTTCCTGCCGCTCCTGTTCTTGAAGCT GGCGTTCCCCAGCAGCAAAGCCCTCTGGATCTGACCAGA GAGCCTCAGCTGGAACCTGGCGAACAGTCTCAAGTGGCC CACGGCACAAATGGCATCCATGTGACAGGCGGCAGCAT GACCATCACCGGCAACATCTACATCTACAACGGCCCCGTGCTCGGCGGACCTCCTGGACCAGGCGATCTTCCTGCTAC ACCCGAGCCTCCATATCCTATTCCAGAGGAAGGCGACCC TGGACCACCTGGCCTTTCTACACCTCACCAAGAGGATGG CAAGGCCTGGCACCTCGCCGAGACAGAACATTGTGGCGC TACCCCTAGCAACAGGGGCCCCAGAAATCAGTTCATCAC CCACGACTGA Truncated LNGFR MGAGATGRAMDGPRLLLLLLLGVSLGGA signal peptide (amino acid) Truncated LNGFR ATGGGAGCTGGTGCTACAGGCAGAGCCATGGATGGACCT signal peptide AGACTGCTGCTGCTCCTGCTGCTTGGAGTGTCTCTTGGCG (nucleotide) GAGCC Truncated LNGFR KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCL (amino acid) DSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCR CAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEE CPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGV VTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAF KR Truncated LNGFR AAAGAGGCCTGTCCTACCGGCCTGTATACACACTCTGGC (nucleotide) GAGTGCTGCAAGGCCTGCAATCTTGGAGAAGGCGTGGCA CAGCCTTGCGGCGCTAATCAGACAGTGTGCGAGCCTTGC CTGGACAGCGTGACCTTTAGCGACGTGGTGTCTGCCACC GAGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAG AGCATGTCTGCCCCTTGTGTGGAAGCCGACGATGCCGTG TGTAGATGCGCCTACGGCTACTACCAGGACGAGACAACA GGCAGATGCGAGGCCTGTAGAGTGTGTGAAGCCGGCTCT GGACTGGTGTTCAGCTGCCAAGACAAGCAGAACACCGTG TGCGAGGAATGCCCCGATGGCACCTATAGCGACGAGGCC AACCATGTGGATCCCTGCCTGCCTTGTACTGTGTGCGAA GATACCGAGCGGCAGCTGCGCGAGTGTACAAGATGGGC TGATGCCGAGTGCGAAGAGATCCCCGGCAGATGGATCAC CAGAAGCACACCTCCAGAGGGCAGCGATAGCACAGCCC CTTCTACACAAGAGCCCGAGGCTCCTCCTGAGCAGGATC TGATTGCCTCTACAGTGGCCGGCGTGGTCACAACAGTGA TGGGATCTTCTCAGCCCGTGGTCACCAGAGGCACCACCG ACAATCTGATCCCCGTGTACTGTAGCATCCTGGCCGCCG TGGTTGTGGGACTCGTGGCCTATATCGCCTTCAAGAGAT GATAA Full length CD27 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPFull length CD27 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CT CD27 extracellular ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRK domain AAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITA (amino acid) NAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPH PQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWP PQRSLCSSDFIR CD27 extracellular GCTACACCAGCTCCTAAGAGCTGCCCCGAGAGACACTAT domain TGGGCCCAGGGCAAGCTGTGCTGCCAGATGTGTGAACCT (nucleotide) GGCACCTTCCTGGTCAAGGACTGCGACCAGCACAGAAAG GCCGCTCAGTGCGATCCTTGTATCCCCGGCGTGTCCTTCT CTCCCGACCACCACACAAGACCTCACTGCGAGAGCTGCA GACACTGCAATTCTGGACTGCTCGTGCGGAACTGCACCA TCACAGCCAATGCCGAGTGCGCCTGCAGAAATGGCTGGC AGTGCCGGGACAAAGAATGTACCGAGTGCGACCCTCTGC CTAATCCTAGCCTGACCGCCAGAAGCAGCCAGGCTTTGT CTCCTCATCCTCAGCCTACACATCTGCCCTACGTGTCCGA GATGCTGGAAGCCAGAACAGCCGGCCATATGCAGACCCT GGCCGACTTTAGACAGCTGCCCGCCAGAACACTGAGCAC CCATTGGCCTCCACAGAGAAGCCTGTGCAGCAGCGACTT CATCCGG CD27 ILVIFSGMFLVFTLAGALFLH transmembrane domain (amino acid)CD27 ATCCTCGTGATCTTCAGCGGCATGTTCCTGGTGTTCACAC transmembrane TGGCTGGCGCCCTGTTTCTGCAC domain (nucleotide) CD27 cytoplasmic QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKP domain EPACSP (amino acid) CD27 cytoplasmic CAGCGGAGAAAGTACAGAAGCAACAAGGGCGAGAGCCC domain CGTGGAACCTGCCGAGCCTTGTCACTACAGCTGTCCCAG (nucleotide) AGAGGAAGAGGGCAGCACAATCCCCATCCAAGAGGACT ACAGAAAGCCCGAGCCTGCCTGCTCTCCT Truncated CD27 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLH Truncated CD27 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCAC PL648 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPM VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQH DFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK PL648 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCCGCGCGAAGCGATCAGGCAGCGGGGCGACAAAT TTCAGCCTTCTGAAACAAGCAGGCGACGTGGAAGAAAA CCCCGGTCCAATGGTGAGCAAGGGCGAGGAGCTGTTCAC CGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGT AAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGG GCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCT GCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCG TGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCT ACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGG GCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTA TATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGC AGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCA CCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGG ATCACTCTCGGCATGGACGAGCTGTACAAGTAA PL1090 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPML LLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIK HFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKE ITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVS LNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTS GQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVS CRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQA MNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTL VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSI ATGMVGALLLLLVVALGIGLFM PL1090 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAGAAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCCGCGCGAAGCGATCAGGCAGCGGGGCGACAAAT TTCAGCCTTCTGAAACAAGCAGGCGACGTGGAAGAAAA CCCCGGTCCAatgcttctcctggtgacaagccttctgctctgtgagttaccacacccag cattcctcctgatcccacgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactct ccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatc ctgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactgg atattctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaacagg acggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtca gttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataag tgatggagatgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaa aactgtttgggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaacagctgca aggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggcccggagcc cagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaac cttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccaga gtgcctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagt gtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggaga aaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaa actgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgggcctaagatc ccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccctggggatc ggcctcttcatgTGATAA PL1093 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPM GRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNN GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ VTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS PL1093 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCCGCGCGAAGCGATCAGGCAGCGGGGCGACAAAT TTCAGCCTTCTGAAACAAGCAGGCGACGTGGAAGAAAA CCCCGGTCCAatgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcc tgtggacgcgtatcgccagcacgatcccaccgcacgttcagaagtcggttaataacgacatga tagtcactgacaacaacggtgcagtcaagtttccacaactgtgtaaattttgtgatgtgagattttc cacctgtgacaaccagaaatcctgcatgagcaactgcagcatcacctccatctgtgagaagccacaggaagtctgtgtggctgtatggagaaagaatgacgagaacataacactagagacagtttg ccatgaccccaagctcccctaccatgactttattctggaagatgctgcttctccaaagtgcattat gaaggaaaaaaaaaagcctggtgagactttcttcatgtgttcctgtagctctgatgagtgcaatg acaacatcatcttctcagaagaatataacaccagcaatcctgacttgttgctagtcatatttcaagt gacaggcatcagcctcctgccaccactgggagttgccatatctgtcatcatcatcttctactgct accgcgttaaccggcagcagaagctgagttcatagtaa PL1106 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPML LPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQT CRDQEKEYYEPQHRICCSRCPPGTYVSAKCSRIRDTVCATC AENSYNEHWNYLTICQLCRPCDPVMGLEEIAPCTSKRKTQC RCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGN NHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTA QSDTTCKNPLEPLPPEMSGTMLMLAVLLPLAFFLLLATVFS CIWKSHPSLCRKLGSLLKRRPQGEGPNPVAGSWEPPKAHPY FPDLVQPLLPISGDVSPVSTGLPAAPVLEAGVPQQQSPLDLT REPQLEPGEQSQVAHGTNGIHVTGGSMTITGNIYIYNGPVL GGPPGPGDLPATPEPPYPIPEEGDPGPPGLSTPHQEDGKAW HLAETEHCGATPSNRGPRNQFITHD PL1106 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCCGCGCGAAGCGATCAGGCAGCGGGGCGACAAAT TTCAGCCTTCTGAAACAAGCAGGCGACGTGGAAGAAAA CCCCGGTCCAATGCTGCTGCCTTGGGCTACATCTGCTCCA GGACTTGCTTGGGGACCTCTGGTGCTGGGACTGTTTGGA CTGCTGGCTGCCTCTCAGCCTCAAGCCGTTCCTCCATACG CCAGCGAGAACCAGACCTGCCGGGACCAAGAGAAAGAG TACTACGAGCCCCAGCACCGGATCTGCTGTAGCAGATGT CCTCCTGGCACATACGTGTCCGCCAAGTGCAGCAGAATC CGGGATACCGTGTGTGCCACCTGTGCCGAGAACAGCTAC AACGAGCACTGGAACTACCTGACCATCTGCCAGCTGTGC AGGCCCTGCGATCCTGTGATGGGACTCGAGGAAATCGCC CCTTGCACCAGCAAGCGCAAGACCCAGTGCAGATGCCA GCCAGGCATGTTCTGTGCCGCCTGGGCTCTCGAATGTAC CCACTGTGAACTGCTGAGCGACTGCCCTCCTGGAACAGA GGCCGAGCTGAAGGATGAAGTCGGCAAGGGCAACAACC ACTGCGTGCCATGTAAAGCCGGCCACTTCCAGAACACAA GCAGCCCTAGTGCCAGGTGCCAGCCTCACACCAGATGCG AGAATCAGGGCCTCGTGGAAGCTGCCCCTGGAACTGCTC AGAGCGACACCACATGCAAGAACCCTCTGGAACCCCTGC CTCCTGAGATGAGCGGCACAATGCTGATGCTGGCCGTTC TGCTGCCTCTGGCCTTCTTTCTGCTGCTGGCCACCGTGTT CAGCTGCATCTGGAAGTCTCACCCCAGCCTGTGCAGAAA GCTGGGCAGCCTGCTTAAGAGAAGGCCTCAAGGCGAGG GCCCTAATCCTGTGGCTGGATCTTGGGAGCCTCCTAAGG CTCACCCTTACTTCCCCGATCTGGTGCAGCCTCTGCTGCC AATCAGCGGAGATGTGTCCCCTGTGTCTACCGGACTTCC TGCCGCTCCTGTTCTTGAAGCTGGCGTTCCCCAGCAGCA AAGCCCTCTGGATCTGACCAGAGAGCCTCAGCTGGAACC TGGCGAACAGTCTCAAGTGGCCCACGGCACAAATGGCAT CCATGTGACAGGCGGCAGCATGACCATCACCGGCAACAT CTACATCTACAACGGCCCCGTGCTCGGCGGACCTCCTGGACCAGGCGATCTTCCTGCTACACCCGAGCCTCCATATCC TATTCCAGAGGAAGGCGACCCTGGACCACCTGGCCTTTC TACACCTCACCAAGAGGATGGCAAGGCCTGGCACCTCGC CGAGACAGAACATTGTGGCGCTACCCCTAGCAACAGGG GCCCCAGAAATCAGTTCATCACCCACGACTGA PL1107 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR PL1107 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCTAA PL1110 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPM GAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSG ECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATE PCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTG RCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANH VDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTP PEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPV VTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR PL1110 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCCGCGCGAAGCGATCAGGCAGCGGGGCGACAAAT TTCAGCCTTCTGAAACAAGCAGGCGACGTGGAAGAAAA CCCCGGTCCAATGGGAGCTGGTGCTACAGGCAGAGCCAT GGATGGACCTAGACTGCTGCTGCTCCTGCTGCTTGGAGT GTCTCTTGGCGGAGCCAAAGAGGCCTGTCCTACCGGCCT GTATACACACTCTGGCGAGTGCTGCAAGGCCTGCAATCT TGGAGAAGGCGTGGCACAGCCTTGCGGCGCTAATCAGAC AGTGTGCGAGCCTTGCCTGGACAGCGTGACCTTTAGCGA CGTGGTGTCTGCCACCGAGCCATGCAAGCCTTGTACCGA GTGTGTGGGCCTGCAGAGCATGTCTGCCCCTTGTGTGGA AGCCGACGATGCCGTGTGTAGATGCGCCTACGGCTACTA CCAGGACGAGACAACAGGCAGATGCGAGGCCTGTAGAG TGTGTGAAGCCGGCTCTGGACTGGTGTTCAGCTGCCAAG ACAAGCAGAACACCGTGTGCGAGGAATGCCCCGATGGC ACCTATAGCGACGAGGCCAACCATGTGGATCCCTGCCTG CCTTGTACTGTGTGCGAAGATACCGAGCGGCAGCTGCGC GAGTGTACAAGATGGGCTGATGCCGAGTGCGAAGAGAT CCCCGGCAGATGGATCACCAGAAGCACACCTCCAGAGG GCAGCGATAGCACAGCCCCTTCTACACAAGAGCCCGAG GCTCCTCCTGAGCAGGATCTGATTGCCTCTACAGTGGCC GGCGTGGTCACAACAGTGATGGGATCTTCTCAGCCCGTG GTCACCAGAGGCACCACCGACAATCTGATCCCCGTGTAC TGTAGCATCCTGGCCGCCGTGGTTGTGGGACTCGTGGCC TATATCGCCTTCAAGAGATGATAA PL1179 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGRGLLR GLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLP PLGVAISVIIIFYCYRVNRQQKLSS PL1179 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCGGCAGCGGGGCGACAAATTTCAGCCTTCTGAAAC AAGCAGGCGACGTGGAAGAAAACCCCGGTCCAatgggtcggg ggctgctcaggggcctgtggccgctgcacatcgtcctgtggacgcgtatcgccagcacgatc ccaccgcacgttcagaagtcggttaataacgacatgatagtcactgacaacaacggtgcagtc aagtttccacaactgtgtaaattttgtgatgtgagattttccacctgtgacaaccagaaatcctgca tgagcaactgcagcatcacctccatctgtgagaagccacaggaagtctgtgtggctgtatgga gaaagaatgacgagaacataacactagagacagtttgccatgaccccaagctcccctaccat gactttattctggaagatgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtga gactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatcatcttctcagaagaatat aacaccagcaatcctgacttgttgctagtcatatttcaagtgacaggcatcagcctcctgccacc actgggagttgccatatctgtcatcatcatcttctactgctaccgcgttaaccggcagcagaagc tgagttcatagtaaPL1099 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTI PIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGAGAT GRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKA CNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPC TECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEAC RVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCL PCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDS TAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTT DNLIPVYCSILAAVVVGLVAYIAFKR PL1099 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACCAGCGGAGAAAGTACAGAAGCAACAA GGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTA CAGCTGTCCCAGAGAGGAAGAGGGCAGCACAATCCCCA TCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTC CTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC CTCGCGGCAGCGGGGCGACAAATTTCAGCCTTCTGAAAC AAGCAGGCGACGTGGAAGAAAACCCCGGTCCAATGGGA GCTGGTGCTACAGGCAGAGCCATGGATGGACCTAGACTG CTGCTGCTCCTGCTGCTTGGAGTGTCTCTTGGCGGAGCCA AAGAGGCCTGTCCTACCGGCCTGTATACACACTCTGGCG AGTGCTGCAAGGCCTGCAATCTTGGAGAAGGCGTGGCAC AGCCTTGCGGCGCTAATCAGACAGTGTGCGAGCCTTGCC TGGACAGCGTGACCTTTAGCGACGTGGTGTCTGCCACCG AGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAGA GCATGTCTGCCCCTTGTGTGGAAGCCGACGATGCCGTGT GTAGATGCGCCTACGGCTACTACCAGGACGAGACAACA GGCAGATGCGAGGCCTGTAGAGTGTGTGAAGCCGGCTCT GGACTGGTGTTCAGCTGCCAAGACAAGCAGAACACCGTG TGCGAGGAATGCCCCGATGGCACCTATAGCGACGAGGCC AACCATGTGGATCCCTGCCTGCCTTGTACTGTGTGCGAA GATACCGAGCGGCAGCTGCGCGAGTGTACAAGATGGGC TGATGCCGAGTGCGAAGAGATCCCCGGCAGATGGATCAC CAGAAGCACACCTCCAGAGGGCAGCGATAGCACAGCCC CTTCTACACAAGAGCCCGAGGCTCCTCCTGAGCAGGATC TGATTGCCTCTACAGTGGCCGGCGTGGTCACAACAGTGA TGGGATCTTCTCAGCCCGTGGTCACCAGAGGCACCACCG ACAATCTGATCCCCGTGTACTGTAGCATCCTGGCCGCCG TGGTTGTGGGACTCGTGGCCTATATCGCCTTCAAGAGAT GATAA PL1091 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPRRAKRSGSGATNFSLLKQAGDVEENPGPMLLLVTSLL LCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCT SISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLI QAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKI ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVS RGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITC TGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGM VGALLLLLVVALGIGLFM PL1091 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGAGACCAGTACAAACTACT CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCA GGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGA GTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGA GATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCCGCGCGAAGCGATCAGGCAGCG GGGCGACAAATTTCAGCCTTCTGAAACAAGCAGGCGACG TGGAAGAAAACCCCGGTCCAatgcttctcctggtgacaagccttctgctctgt gagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggtattggtg aatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatca gtggcgatctccacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctg gatccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattcagg cttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcagg accaagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacg ctccctcaaggagataagtgatggagatgtgataatttcaggaaacaaaaatttgtgctatgcaa atacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagcaaca gaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgaggg ctgctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaa tgcgtggacaagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagtg catacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgggga ccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgt gccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaa cgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctgg tggtggccctggggatcggcctcttcatgTGATAA PL1094 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRP HCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTE CDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHM QTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVF TLAGALFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPRRAKRSGSGATNFSLLKQAGDVEENPGPMVSKGEEL FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC TTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRH NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKD PNEKRDHMVLLEFVTAAGITLGMDELYK PL1094 ATGGCCAGACCTCATCCTTGGTGGCTGTGTGTGCTGGGC (nucleotide) ACACTCGTTGGCCTGTCTGCTACACCAGCTCCTAAGAGC TGCCCCGAGAGACACTATTGGGCCCAGGGCAAGCTGTGC TGCCAGATGTGTGAACCTGGCACCTTCCTGGTCAAGGAC TGCGACCAGCACAGAAAGGCCGCTCAGTGCGATCCTTGT ATCCCCGGCGTGTCCTTCTCTCCCGACCACCACACAAGA CCTCACTGCGAGAGCTGCAGACACTGCAATTCTGGACTG CTCGTGCGGAACTGCACCATCACAGCCAATGCCGAGTGC GCCTGCAGAAATGGCTGGCAGTGCCGGGACAAAGAATG TACCGAGTGCGACCCTCTGCCTAATCCTAGCCTGACCGC CAGAAGCAGCCAGGCTTTGTCTCCTCATCCTCAGCCTAC ACATCTGCCCTACGTGTCCGAGATGCTGGAAGCCAGAAC AGCCGGCCATATGCAGACCCTGGCCGACTTTAGACAGCT GCCCGCCAGAACACTGAGCACCCATTGGCCTCCACAGAG AAGCCTGTGCAGCAGCGACTTCATCCGGATCCTCGTGAT CTTCAGCGGCATGTTCCTGGTGTTCACACTGGCTGGCGCC CTGTTTCTGCACAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGAGACCAGTACAAACTACT CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCA GGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGA GTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGA GATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCCGCGCGAAGCGATCAGGCAGCG GGGCGACAAATTTCAGCCTTCTGAAACAAGCAGGCGACG TGGAAGAAAACCCCGGTCCAATGGTGAGCAAGGGCGAG GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCT GAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTG GCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTG CTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCG CACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCG CGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGC AACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAAC ACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCC AACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG ACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC AAGTAA PL1108 MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLC (amino acid) CQ...

Claims

CLAIMS What is claimed is:

1. An isolated nucleic acid comprising a nucleotide sequence encoding a dual / multispecific chimeric antigen receptor (CAR) comprising: a first chimeric antigen receptor (CAR), wherein the first CAR comprises a full length CD27 receptor, a first costimulatory domain, and a first intracellular signaling domain; a cleavage sequence; and a second CAR, wherein the second CAR comprises an affinity binding domain that binds to at least one disease-associated antigen of interest, a second costimulatory domain, and a second intracellular signaling domain.

2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide comprising, from C-terminus to N-terminus, the first CAR, the cleavage sequence, and the second CAR.

3. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide comprising, from C-terminus to N-terminus, the second CAR, the cleavage sequence, and the first CAR.

4. The isolated nucleic acid of any one of claims 1-3, wherein the cleavage sequence is a 2A self-cleaving sequence selected from T2A, P2A, E2A, and F2A self-cleaving sequences.

5. The isolated nucleic acid sequence of claim 4, wherein the 2A self-cleaving sequence is a P2A self-cleaving sequence; optionally wherein the P2A self-cleaving sequence encoded by the isolated nucleic acid has an amino acid sequence as set forth in SEQ ID NO: 8, 68, or 69.

6. The isolated nucleic acid of any preceding claim, wherein the second CAR further comprises a hinge domain; optionally wherein the hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or a receptor-derived hinge; preferably wherein the receptor-derived hinge is a CD8 alpha hinge domain, optionally wherein the CD8 alpha hinge domain comprises an amino acid sequence set forth as SEQ ID NO: 97.

7. The isolated nucleic acid of any preceding claim, wherein the second CAR further comprises a transmembrane (TM) domain; optionally wherein the TM domain comprises a TM domain of CD8, preferably wherein the CD8 TM domain is a TM domain of CD8 alpha; optionally wherein the TM domain comprises the amino acid sequence set forth as SEQ ID NO:

99.

8. The isolated nucleic acid of any preceding claim, wherein the first and / or second costimulatory domain is 4-1BB; optionally wherein the 4-1BB costimulatory domain comprises an amino acid sequence set forth as SEQ ID NO:

4.

9. The isolated nucleic acid of any preceding claim, wherein the first and / or second intracellular signaling domain is selected from CD3ζ, DAP12, LFA-1, and repeat (2-5) DAP10 YINM motifs; preferably wherein the first and / or second intracellular signaling domain is CD3ζ.

10. The isolated nucleic acid of any preceding claim, wherein the second CAR is a tandem CAR.

11. The isolated nucleic acid of any preceding claim, wherein the at least one disease- associated antigen is a tumor antigen, an autoantigen, or a pathogenic antigen.

12. The isolated nucleic acid of claim 11, wherein the disease-associated antigen of interest is selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L.

13. The isolated nucleic acid of claim 11, wherein the disease-associated antigen of interest is B cell antigen selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB.

14. The isolated nucleic acid of claim 11, wherein the disease-associated antigen of interest is plasma cell antigen selected from the group comprising or consisting of CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB.

15. The isolated nucleic acid of any preceding claim, wherein the isolated nucleic acid further encodes one or more additional polypeptides.

16. The isolated nucleic acid of claim 2, wherein the second CAR comprises an affinity binding domain that binds to CD19; optionally wherein the isolated nucleic acid comprises the sequence set forth in SEQ ID NO:

117.

17. The isolated nucleic acid of claim 3, wherein the second CAR comprises an affinity binding domain that binds to CD19; optionally wherein the isolated nucleic acid comprises the sequence set forth in SEQ ID NO:

121.

18. A recombinant expression vector comprising the nucleic acid of any one of claims 1-17.

19. An isolated host cell comprising the recombinant expression vector of claim 18.

20. A multispecific CAR T cell having increased persistence comprising: a first chimeric antigen receptor (CAR), wherein the first CAR comprises full length CD27 receptor (SEQ ID NO: 32), a first costimulatory domain, and a first intracellular signaling domain; and a second CAR, wherein the second CAR comprises an affinity binding domain that binds to at least one disease-associated antigen of interest, a second costimulatory domain, and a second intracellular signaling domain, wherein the multispecific CAR T cell functionally expresses the CD27 receptor and the affinity binding domain on surface of the T cell.

21. The multispecific CAR T cell of claim 20, wherein the second CAR further comprises a hinge domain; optionally wherein the hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or a receptor-derived hinge; preferably wherein the receptor-derived hinge is a CD8 alpha hingedomain, optionally wherein the CD8 alpha hinge domain comprises an amino acid sequence set forth as SEQ ID NO:

97.

22. The multispecific CAR T cell of any one of claims 20-21, wherein the second CAR further comprises a transmembrane (TM) domain; optionally wherein the TM domain comprises a TM domain of CD8, preferably wherein the CD8 TM domain is a TM domain of CD8 alpha; optionally wherein the TM domain comprises the amino acid sequence set forth as SEQ ID NO:

99.

23. The multispecific CAR T cell of any one of claims 20-22, wherein the first and / or second costimulatory domain is 4-1BB; optionally wherein the 4-1BB costimulatory domain comprises an amino acid sequence set forth as SEQ ID NO:

4.

24. The multispecific CAR T cell of any one of claims 20-23, wherein the first and / or second intracellular signaling domain is selected from CD3ζ, DAP12, LFA-1, and repeat (2-5) DAP10 YINM motifs; preferably wherein the first and / or second intracellular signaling domain is CD3ζ.

25. The multispecific CAR T cell of any one of claims 20-24, wherein the second CAR is a tandem CAR.

26. The multispecific CAR T cell of any one of claims 20-25, wherein the at least one disease-associated antigen of interest is a tumor antigen, an autoantigen, or a pathogenic antigen.

27. The multispecific CAR T cell of claim 26, wherein the disease-associated antigen of interest is selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. 28 The multispecific CAR T cell of claim 27, wherein the disease-associated antigen of interest is a B cell antigen selected from the group comprising or consisting of CD19, CD20,CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB.

29. The multispecific CAR T cell of claim 27, wherein the disease-associated antigen of interest is a plasma cell antigen selected from the group comprising or consisting of CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB.

30. The multispecific CAR T cell of any one of claims 20-29, wherein the isolated nucleic acid further encodes one or more additional polypeptides.

31. The multispecific CAR T cell of claim 20, wherein the second CAR comprises an affinity binding domain that binds to CD19; optionally wherein the first CAR comprises the amino acid sequence set forth in SEQ ID NO: 138 and the second CAR comprises the amino acid sequence set forth in SEQ ID NO:

139.

32. The multispecific CAR T cell of any one of claims 20-31 wherein the T cell is a γδ T cell, preferably wherein the γδ T cell is a δ1 γδ T cell.

33. A pharmaceutical composition comprising a plurality of multispecific CAR T cells according to any one of claims 20-32, wherein the plurality comprises at least about 107T cells, preferably from about 108T cells to about 1011T cells; optionally further comprising a pharmaceutically acceptable excipient.

34. The pharmaceutical composition according to claim 33, wherein the plurality comprises a composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2- γδ T cells, most preferably δ1 γδ T cells.

35. A method of treating an autoimmune disease in a subject in need thereof, comprising: administering a therapeutically effective amount of multispecific CAR T cells according to any one of claims 20-32, or a pharmaceutical composition according to claim 33 or 34.

36. The method of claim 35, comprising performing a plurality of administrations of the multispecific CAR T cells, wherein the interval between the plurality of administrations is at least about a week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or wherein the plurality of administrations is no more than once every 6 or 12 months.

37. The method of claim 35 or 36, the autoimmune disease is lupus, lupus nephritis, systemic sclerosis, idiopathic inflammatory myopathies, or myasthenia gravis.

38. The method of claim 37, wherein the autoimmune disease is lupus.

39. A method of treating cancer in a subject in need thereof, comprising: administering a therapeutically effective amount of multispecific CAR T cells according to any one of claims 20-32, or a pharmaceutical composition according to claim 33 or 34, wherein the cancer comprises hematological and / or solid tumor cells that exhibit cell surface expression of CD70.

40. The method of claim 39, wherein the method comprises performing a plurality of administrations of the multispecific CAR T cells, wherein the interval between the plurality of administrations is at least about a week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or wherein the plurality of administrations is no more than once every 6 or 12 months.

41. Use of the multispecific CART cell of any one of claims 20-32, or the pharmaceutical composition of claim 33 or 34, in the manufacture of a medicament for the treatment or prevention of autoimmune disease or cancer.

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