Chimeric antigen receptor t cells (car-t) for treatment of cancer

Abstract

To provide improved CAR-based immunotherapeutic drugs utilizing genome-edited and mono, dual, and tandem chimeric antigen receptor (CAR) constructs for targeting cancer.SOLUTION: There are provided, genome-edited chimeric antigen receptor T cells (CAR-T), which can be derived from cytotoxic T cells, a viral-specific cytotoxic T cell, memory T cells, or gamma delta (γδ) T cells, and comprise one or more chimeric antigen receptors (CARs) targeting one or more antigens, where the CAR-T cells are deficient in one or more antigens to which the one or more CARs specifically bind. In particular, the present disclosure provides engineered mono, dual, and tandem chimeric antigen receptor (CAR)-bearing T cells (CAR-T) and methods of immunotherapy for the treatment of cancer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 799,513, filed January 31, 2019, and U.S. Provisional Patent Application No. 62 / 678,878, filed May 31, 2018, the disclosures of which are incorporated by reference herein as if set forth in their entireties.

[0002] Disclosed herein are genome-edited chimeric antigen receptor T cells (CAR-T) and methods of use thereof in immunotherapy. In particular, the disclosure relates to T cells that can be genetically modified to express one or more chimeric antigen receptors (CARs) and methods of use thereof in the treatment of cancer. [Background technology]

[0003] T cells, a type of lymphocyte, play a central role in cell-mediated immunity. T cells are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of the T cell receptor (TCR) on the cell surface. CD4 + T helper cells (T), also called T or CD4 T cells H ) express the CD4 glycoprotein on their surface. Helper T cells become activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, these cells proliferate rapidly and secrete cytokines that regulate the immune response. CD8 + Cytotoxic T cells (T cells), also known as T cells or CD8 T cells C ) express the CD8 glycoprotein on their cell surface. +T cells are activated when exposed to peptide antigens presented by MHC class I molecules. A subtype of T cells, memory T cells, respond to their cognate antigens over time, providing an immune mechanism with "memory" against past infections and / or tumor cells. Gamma delta (γδ) T cells are the epitome of "unconventional" T cells and represent a relatively small subpopulation of T cells in peripheral blood. Gamma delta (γδ) T cells are defined by their expression of a heterodimeric T cell receptor (TCR) composed of γ and δ chains. By this definition, gamma delta (γδ) T cells are CD4 + Helper T cells and CD8 + Virus-specific cytotoxic T lymphocytes are distinct from cytotoxic T cells. They are T cells that are reactive against viral antigens, particularly Epstein-Barr virus (EBV) and cytomegalovirus (CMV).

[0004] The T cells described herein may be genetically modified to express a chimeric antigen receptor (CAR), a fusion protein composed of an antigen recognition portion and a T cell activation domain. T cells expressing a CAR can recognize a specific protein, i.e., an antigen, on a tumor cell. These T cells expressing a CAR may be expanded in the laboratory and then infused into a patient.

[0005] Clinical trials have shown high response rates following infusion of anti-CD19 CARs in patients with B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL) and B-cell precursor acute lymphoblastic leukemia (ALL), leading to two FDA-approved therapies, Yescarta™ (axicabtagene ciloleucel, Kite Pharma / Gilead) and Kymriah™ (tisagenlecleucel, Novartis). Despite these successes, the development of CAR-T cell therapeutics for T-cell malignancies has proven to have unsolved problems, in part due to the common expression of target antigens between malignant and effector T cells. Among the most common challenges are (1) the antigen target(s) of the chimeric antigen receptor(s), (2) CAR design, i.e., mono-CAR, dual CAR, tandem CAR, and (3) tumor heterogeneity, especially differences in surface expression of tumor antigens. As a result, there remains a need for improved chimeric antigen receptor (CAR)-based immunotherapeutics utilizing genome editing and constructs of mono-, dual-, and tandem CARs to more effectively, safely, and efficiently target cancers, including T cell-related malignancies. [Brief description of the drawings]

[0006] [Figure 1] A schematic diagram of dual CAR-T cells (dCAR-T cells) is shown.

[0007] [Diagram 2] 1 shows a schematic diagram of tandem CAR-T cells (tCAR-T cells).

[0008] [Diagram 3] Schematic diagrams of dual and tandem CAR constructs are shown.

[0009] [Figure 4] Schematic diagram of tandem targeted CAR construct.

[0010] [Diagram 5]Shows the purity of the CAR-T construct without physical depletion of CD3+ or CD2+ CAR-T cells. As shown by FACS analysis, there is high purity of CD3- and CD2- CAR-T cells without the need for magnetic depletion of CD3+ cells. Representative FACS plots show FITC staining of CD3 (y-axis) and CD2 (x-axis). Clone 5 (top) and clone 6 (bottom) are shown.

[0011] [Figure 6] Shows the purity of the CAR-T construct without physical depletion of CD3+ or CD2+ CAR-T cells. As shown by FACS analysis, there is high purity of CD3- and CD2- CAR-T cells without the need for magnetic depletion selection of CD3+ cells. Representative FACS plots show FITC staining of CD3 (y-axis) and CD2 (x-axis). Clone 7 (top) and Clone 8 (bottom) are shown.

[0012] [Figure 7] The purity of the CAR-T construct is shown without physical depletion of CD3+ or CD2+ CAR-T cells. As shown by FACS analysis, there is high purity of CD3- and CD2- CAR-T cells without the need for magnetic depletion selection of CD3+ cells. Representative FACS plots show FITC staining of CD3 (y-axis) and CD2 (x-axis). Clone 13 (top) and Clone 14 (bottom) are shown.

[0013] [Figure 8] The purity of the CAR-T construct is shown without physical depletion of CD3+ or CD2+ CAR-T cells. As shown by FACS analysis, there is high purity of CD3- and CD2- CAR-T cells without the need for magnetic depletion selection of CD3+ cells. Representative FACS plots show FITC staining of CD3 (y-axis) and CD2 (x-axis). Clone 15 (top) and Clone 16 (bottom) are shown.

[0014] [Figure 9A] Tumor cell killing of tandem CD2-CD3 CAR-T clone 5 (top) and tandem CD2-CD3 CAR-T clone 6 (bottom) is shown, and the legend indicates the effector to target cell ratio (E:T ratio).

[0015] [Figure 9B] Tumor cell killing of tandem CD2-CD3 CAR-T clone 7 (top) and tandem CD2-CD3 CAR-T clone 8 (bottom) is shown, with the legend indicating the effector to target cell ratio (E:T ratio).

[0016] [Figure 9C] Tumor cell killing of tandem CD2-CD3 CAR-T clone 13 (top) and tandem CD2-CD3 CAR-T clone 14 (bottom) is shown, with the legend indicating the effector to target cell ratio (E:T ratio).

[0017] [Figure 9D] Tumor cell killing of tandem CD2-CD3 CAR-T clone 15 (top) and tandem CD2-CD3 CAR-T clone 16 (bottom) is shown, with the legend indicating the effector to target cell ratio (E:T ratio).

[0018] [Figure 10A] Schematic diagram of BCMA CAR construct for targeting BCMA and transducing T cells.

[0019] [Figure 10B] Figure 1 shows tumor cell killing of BCMA-CAR-T cells in a 51Cr release assay. Efficient killing of BCMA-CAR-T cells was observed at multiple effector to target (E:T) ratios. Non-transduced activated T cells and CD19-CAR-T cells were used as negative controls and did not induce killing of MM.1S-CG cells.

[0020] [Figure 10C] Showing the in vivo efficacy of BCMA CAR-T cells. All seven mice treated with BCMA CAR-T survived for nearly 150 days or longer, compared to controls that died around day 50.

[0021] [Figure 10D] They show that time-lapse bioluminescence imaging (BLI) measuring photon flux revealed a strong reduction in signal to background levels that never increased over the duration of the experiment in mice treated with BCMA CAR-T cells.

[0022] [Figure 11A] A schematic diagram of the CS1-CAR construct that targets CS1 and transduces T cells is shown.

[0023] [Figure 11B] Showing the in vivo efficacy of CS1-CAR-T cells. As a way to test the specificity of CS1-CAR-T cells, mice were implanted with MM.1S-CG cells and MM.1S-CG cells lacking CS1 (MM.1S-CGΔCS1, using Cas9 / CRISPR technology). All mice treated with CS1-CAR-T cells (n=10) survived >90 days, while the median survival of CD19 control mice (n=8) was 43 days.

[0024] [Figure 11C] We show that time-lapse bioluminescence imaging (BLI) showed that mice treated with CS1-CAR-T cells showed a 3 log reduction in photon flux and elimination of bone marrow tumors (Experiment 1-Experiment 3).

[0025] [Figure 12A] Schematic diagram of mono (CD19, CS1) and tandem (BCMA-CS1) constructs.

[0026] [Figure 12B]FACS analysis shows that Jurkat cells expressing CD19 CAR did not bind either BCMA or CS1 protein (lower left quadrant of each plot). Jurkat cells expressing BCMA CAR protein bound to BCMA protein (upper left quadrant of each plot). Jurkat cells expressing CS1 CAR protein bound to CS1 protein (lower right quadrant of each plot). Jurkat cells expressing tandem BCMA-CS1 CAR protein bound to both recombinant proteins (upper right quadrant of each plot), indicating expression of both scFvs.

[0027] [Figure 12C] Showing the in vitro efficacy of single and tandem CAR-T cells using a standard 4-hour chromium release (51Cr) assay. BCMA-CS1 tCAR-T cells killed MM.1S-CG cells with similar efficacy as both single antigen-targeted BCMA and single antigen-targeted CS1 CAR-T cells.

[0028] [Figure 13] 1 shows an efficacy study of CD2*CD3Δ-dCARTΔCD2ΔCD3ε using a xenogeneic model of T-ALL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Disclosed below are detailed embodiments, options, and uses of genetically modified cells, and the use of such cells in immunotherapy and adoptive cell transfer, for example, to treat disease. Accordingly, the following embodiments are provided herein:

[0030] Embodiment 1. A CAR-T cell comprising one or more chimeric antigen receptors (CARs) targeting one or more antigens, wherein the CAR-T cell is missing a subunit of the T cell receptor complex and / or is missing at least one or more antigens to which the one or more CARs specifically bind.

[0031] Embodiment 2. A CAR-T cell comprising one or more chimeric antigen receptors (CARs) that target one or more antigens, wherein the CAR-T cell lacks the one or more antigens to which the one or more CARs specifically bind.

[0032] Embodiment 3. The CAR-T cell of embodiment 1, wherein the subunit of the T cell receptor complex is selected from TCRα, TCRβ, TCRδ, TCRγ, CD3ε, CD3γ, CD3δ, and CD3ζ.

[0033] Embodiment 4. The CAR-T cell of any one of embodiments 1 to 2, wherein the chimeric antigen receptor (CAR) specifically binds to one or more antigens expressed on malignant T cells or myeloma cells.

[0034] Embodiment 5. The CAR-T cell of any one of embodiments 1 to 4, wherein the chimeric antigen receptor (CAR) exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0035] Embodiment 6. The CAR-T cell of any one of embodiments 1 to 4, wherein the chimeric antigen receptor (CAR) exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0036] Embodiment 7. The CAR-T cell of any one of embodiments 1 to 4, wherein the chimeric antigen receptor (CAR) is an amino acid sequence selected from SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0037] Embodiment 8. The CAR-T cell of any one of embodiments 1 to 4, wherein the chimeric antigen receptor(s) specifically bind to one or more antigen(s) selected from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.

[0038] Embodiment 9. The CAR-T cell of any one of embodiments 1 to 5, wherein said chimeric antigen receptor(s) specifically binds to at least one antigen expressed on a malignant T cell.

[0039] Embodiment 10. The CAR-T cell of embodiment 9, wherein the antigen expressed on the malignant T cell is selected from CD2, CD3, CD4, CD5, CD7, TCRβ, and TCRβ.

[0040] Embodiment 11. The CAR-T cell of any one of embodiments 1 to 5, wherein the chimeric antigen receptor specifically binds to at least one antigen expressed on a malignant plasma cell.

[0041] Embodiment 12. The CAR-T cell of embodiment 11, wherein the antigen expressed on malignant plasma cells is selected from BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19.

[0042] Embodiment 13. The CAR-T cell of any one of embodiments 1 to 5, wherein said chimeric antigen receptor(s) specifically binds to at least one antigen expressed on a malignant B cell.

[0043] Embodiment 14. The CAR-T cell of embodiment 13, wherein the antigen expressed on malignant B cells is selected from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.

[0044] Embodiment 15. The CAR-T cell of embodiment 14, wherein the antigen expressed on malignant B cells is selected from CD19 and CD20.

[0045] Embodiment 16. The CAR-T cell of any one of embodiments 1 to 15, further comprising a suicide gene.

[0046] Embodiment 17. The CAR-T cell of any one of embodiments 1 to 16, wherein endogenous T cell receptor-mediated signaling is inhibited in said CAR-T cell.

[0047] Embodiment 18. A CAR-T cell according to any one of embodiments 1 to 17, which does not induce alloreactivity or graft-versus-host disease.

[0048] Embodiment 19. A CAR-T cell according to any one of embodiments 1 to 18, which does not induce sibling killing.

[0049] Embodiment 20. A dual or tandem CAR-T cell according to any one of embodiments 1 to 19.

[0050] Embodiment 21. The CAR(s) is selected from the group consisting of CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD7, CD 2×CD3ε, CD2×CD4, CD2×CD5, CD2×CD7, CD3ε×CD4, CD3ε×CD5, CD3ε×CD7, CD4×CD5, CD4×CD7, CD5×CD7, TRAC×CD2, TRAC×CD3ε, TRAC×CD4, TRAC×CD5, TRAC×CD7, TCRβ×CD2, TCRβ×CD3ε, TCRβ×CD4, TCRβ×CD5, TCRβ×CD7, BCMA×CS1, BCMA× CD19, BCMA×CD38, CS1×CD19, CD19×CD38, APRIL×CS1, APRIL×BCMA, APRIL×CD19, APRIL×CD38, CS1×CD38, CD79A×BC MA, CD79A×CS1, CD79A×CD19, CD79A×CD38, CD79A×CD38, CD79A×APRIL, CD79A×CD79B, CD79B×BCMA, CD79B×CS1, CD7 21. The CAR-T cell of embodiment 20, which specifically binds to two different targets selected from: 9BxCD19, CD79BxCD38, CD79BxAPRIL, CD79BxCD79A, CD138xBCMA, CD138xCS1, CD138xCD19, CD138xCD38, CD138xAPRIL, CD138xCD79A, CD138xCD79B, CD138xBCMA, and CD138xCS1.

[0051] Embodiment 22. The CAR(s) are selected from the group consisting of CD2×CD3ε, CD2×CD4, CD2×CD5, CD2×CD7, CD3ε×CD4, CD3ε×CD5, CD3ε×CD7, CD4×CD5, CD4×CD7, CD5×CD7, TRAC×CD2, TRAC×CD3ε, TRAC×CD4, TRAC×CD5, TRAC×CD7, TCRβ×CD2, TCRβ×CD3ε, TCRβ×CD4, TCRβ×CD7, CD2×CD3ε, CD2×CD4, 22. The CAR-T cell of embodiment 21, which specifically binds to two different targets selected from CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD5, and TCRβxCD7.

[0052]

[0023] Embodiment 23. The CAR(s) is selected from the group consisting of BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, APRILxCD38, CS1xCD38, CD79AxBCMA, CD79AxCS1, CD79AxCD19, CD79AxCD38, CD79AxCD38, CD79AxAPRIL, CD79AxCD79B, CD79BxB 22. The CAR-T cell of embodiment 21, which specifically binds to two different targets selected from: CMA, CD79BxCS1, CD79BxCD19, CD79BxCD38, CD79BxAPRIL, CD79BxCD79A, CD138xBCMA, CD138xCS1, CD138xCD19, CD138xCD38, CD138xAPRIL, CD138xCD79A, CD138xCD79B, CD138xBCMA, and CD138xCS1.

[0053] Embodiment 24. The CAR-T cell of embodiment 21, wherein said CAR(s) specifically bind to two different targets selected from CD123xCD371, CD123xCLEC12A, CD123xCD117, CD123xFLT3, CD123xCD7, CD123xTim3, CD371xCLEC12A, CD371xCD117, CD371xFLT3, CD371xCD7, CD371xTim3, CLEC12AxCD117, CLEC12AxFLT3, CLEC12AxCD7, CLEC12AxTim3, CD117xFLT3, CD117xCD7, CD117xTim3, FLT3xCD7, FLT3xTim3, and CD7xTim3.

[0054] Embodiment 25. A dual CAR-T cell according to any one of embodiments 21 to 24.

[0055] Embodiment 26. A tandem CAR-T cell according to any one of embodiments 21 to 34.

[0056] Embodiment 27. The CAR-T cell of any one of embodiments 1 to 26, further comprising a suicide gene.

[0057] Embodiment 28. A CAR-T cell according to any one of embodiments 1 to 26, wherein endogenous T cell receptor-mediated signaling is inhibited in said CAR-T cell.

[0058] Embodiment 29. A CAR-T cell according to any one of embodiments 1 to 26, which does not induce alloreactivity or graft-versus-host disease.

[0059] Embodiment 30. A CAR-T cell according to any one of embodiments 1 to 26, which does not induce sibling killing.

[0060] Embodiment 31. Dual or tandem chimeric antigen receptors (dCARs or tCARs) targeting two or more plasma cell antigens.

[0061] Embodiment 32. The CAR of embodiment 31, wherein said plasma cell antigen(s) is selected from BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19.

[0062]

[0036] Embodiment 33. The CAR(s) is selected from the group consisting of BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, APRILxCD38, CS1xCD38, CD79AxBCMA, CD79AxCS1, CD79AxCD19, CD79AxCD38, CD79AxCD38, CD79AxAPRIL, CD79AxCD79B, CD79B 33. The CAR of embodiment 32, wherein the CAR specifically binds to two different targets selected from: CD138xBCMA, CD79BxCS1, CD79BxCD19, CD79BxCD38, CD79BxAPRIL, CD79BxCD79A, CD138xBCMA, CD138xCS1, CD138xCD19, CD138xCD38, CD138xAPRIL, CD138xCD79A, CD138xCD79B, CD138xBCMA, and CD138xCS1.

[0063] Embodiment 34. A CAR described in any one of embodiments 31 to 33, which is a dCAR.

[0064] Embodiment 35. A CAR described in any one of embodiments 31 to 33, which is a tCAR.

[0065] Embodiment 36. Dual or tandem chimeric antigen receptors (dCARs or tCARs) targeting two or more leukemia cell antigens.

[0066] Embodiment 37. The CAR of embodiment 36, wherein said plasma cell antigen(s) is selected from CD123, CLEC12A, CD117, FLT3, CD7, and Tim3.

[0067] Embodiment 38. The CAR of embodiment 37, wherein said CAR(s) specifically bind to two different targets selected from CD123xCD371, CD123xCLEC12A, CD123xCD117, CD123xFLT3, CD123xCD7, CD123xTim3, CD371xCLEC12A, CD371xCD117, CD371xFLT3, CD371xCD7, CD371xTim3, CLEC12AxCD117, CLEC12AxFLT3, CLEC12AxCD7, CLEC12AxTim3, CD117xFLT3, CD117xCD7, CD117xTim3, FLT3xCD7, FLT3xTim3, and CD7xTim3.

[0068] Embodiment 39. A CAR described in any one of embodiments 36 to 38, which is a dCAR.

[0069] Embodiment 40. A CAR described in any one of embodiments 36 to 38, which is a tCAR.

[0070] Embodiment 41. Tandem chimeric antigen receptors (tCARs) targeting two or more T cell antigens.

[0071] Embodiment 42. The tCAR of embodiment 41, wherein the T cell antigen is selected from CD5, CD7, CD2, CD4, and CD3.

[0072] Embodiment 43. A tCAR as described in embodiment 42, which targets a pair (i.e., two) of antigens.

[0073] Embodiment 44. The antigen pairs are CD2×CD3ε, CD2×CD4, CD2×CD5, CD2×CD7, CD3ε×CD4, CD3ε×CD5, CD3ε×CD7, CD4×CD5, CD4×CD7, CD5×CD7, TRAC×CD2, TRAC×CD3ε, TRAC×CD4, TRAC×CD5, TRAC×CD7, TCRβ×CD2, TCRβ×CD4, TCRβ×CD7, CD2×CD3ε, C 44. The tCAR of embodiment 43, selected from: D2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD5, and TCRβxCD7.

[0074] Embodiment 45. The tCAR of embodiment 43, wherein the antigen pair is selected from CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, and CD5xCD7.

[0075] Embodiment 46. A tCAR according to any one of embodiments 35 and 40 to 45, wherein the CAR construct is a linear tCAR construct.

[0076] Embodiment 47. The linear tCAR construct comprises a V H 1 and V L The first weight (V H ) chain variable fragment and the first light (V L ) chain variable fragment, and L 2 and V H The second light vehicle, called V2, L ) chain variable fragment and the first heavy (V H ) chain variable fragment, H ) chain variable fragment and the first light (V L ) chain variable fragment is (GGGGS) 2~6 The second light (VL ) chain variable fragment and the first heavy (V H 47. The tCAR of embodiment 46, wherein the tCAR is linked to a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

[0077] Embodiment 48. The linear tCAR construct comprises a V H 2 and V L The first weight (V H ) chain variable fragment and the first light (V L ) chain variable fragment, and H 1 and V L The second light vehicle, called V1, L ) chain variable fragment and the first heavy (V H ) chain variable fragment, H ) chain variable fragment and the first light (V L ) chain variable fragment is (GGGGS) 2~6 The second light (V L ) chain variable fragment and the first heavy (V H 47. The tCAR of embodiment 46, wherein the tCAR is linked to a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

[0078] Embodiment 49. The linear tCAR construct comprises a V L 1 and V H The first light vehicle, called the V L ) chain variable fragment and the first heavy (V H ) chain variable fragment, and H 2 and V L The second heavy (V H ) chain variable fragment and the first light (V L ) chain variable fragment, L ) chain variable fragment and the first heavy (V H ) chain variable fragment is (GGGGS) 2~6 The second moiety (V H ) chain variable fragment and the first light (V L 47. The tCAR of embodiment 46, wherein the tCAR is linked to a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

[0079] Embodiment 50. The linear tCAR construct comprises a V L 2 and V H The first light vehicle, called V2, L ) chain variable fragment and the first heavy (V H ) chain variable fragment, and H 1 and V L The second weight, called 1 (V H ) chain variable fragment and the first light (V L ) chain variable fragment, L ) chain variable fragment and the first heavy (V H ) chain variable fragment is (GGGGS) 2~6 The second moiety (V H ) chain variable fragment and the first light (V L 47. The tCAR of embodiment 46, wherein the tCAR is linked to a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

[0080] Embodiment 51. The tCAR described in embodiment 46, wherein the linear tCAR construct comprises a structure selected from 7-I to 7-XXXII.

[0081] Embodiment 52. A tCAR according to any one of embodiments 35 and 40 to 45, wherein the CAR construct is a hairpin tCAR construct.

[0082] Embodiment 53. The hairpin tCAR construct comprises a V H 1 and V H The first heavy (V H ) chain variable fragment and a second heavy (V H ) chain variable fragment, and L The first light (V12) derived from the second scFv, designated V2 and V13, L ) chain variable fragment and a second light (V L ) chain variable fragment, said first heavy (V H) chain variable fragment and the second heavy (V H ) chain variable fragment is (GGGGS) 2~6 The first light (V) from the second scFv is linked by a linker. L ) chain variable fragment and the second light (V) chain variable fragment derived from the first scFv L 53. The tCAR of embodiment 52, wherein the tCAR is linked to a .) chain variable fragment.

[0083] Embodiment 54. The hairpin tCAR construct comprises a V H 2 and V H A second heavy (V H ) chain variable fragment and the first heavy (V H ) chain variable fragment, and L 1 and V L A first light (V) fragment derived from the first scFv, designated V L ) chain variable fragment and a second light (V) chain variable fragment derived from the second scFv. L ) chain variable fragment, and the second heavy (V H ) chain variable fragment and the first heavy (V H ) chain variable fragment is (GGGGS) 2~6 The first light (V) fragment from the first scFv is linked by a linker. L ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv L 53. The tCAR of embodiment 52, wherein the tCAR is linked to a .) chain variable fragment.

[0084] Embodiment 55. The hairpin tCAR construct comprises a V L 1 and V L The first light (V) derived from the first scFv, designated V L ) chain variable fragment and a second light (V L ) chain variable fragment, and H 2 and V HA first heavy (V H ) chain variable fragment and a second heavy (V L ) chain variable fragment, the first light (V) chain variable fragment derived from the first scFv L ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv L ) chain variable fragment is (GGGGS) 2~6 The first heavy chain (V) from the first scFv is linked by a linker. H ) chain variable fragment and the second heavy (V L 53. The tCAR of embodiment 52, wherein the tCAR is linked to a .) chain variable fragment.

[0085] Embodiment 56. The hairpin tCAR construct comprises a V L 2 and V L A second light (V) antibody derived from a second scFv, designated V L ) chain variable fragment and a first light (V L ) chain variable fragment, and H 1 and V H 2, a first heavy (V H ) chain variable fragment and a second light (V H ) chain variable fragment, and the second light (V) chain variable fragment derived from the second scFv. L ) chain variable fragment and the first light (V) chain variable fragment derived from the first scFv L ) chain variable fragment is (GGGGS) 2~6 The first heavy chain (V) from the first scFv is linked by a linker. H ) chain variable fragment and the second light (V H 53. The tCAR of embodiment 52, wherein the tCAR is linked to a .) chain variable fragment.

[0086] Embodiment 57. The tCAR described in embodiment 52, wherein the hairpin tCAR construct comprises a structure selected from 9-I to 9-XXXII.

[0087] Embodiment 58. A tCAR according to any one of embodiments 35 and 40 to 45, wherein the CAR construct is a hairpin DSB tCAR construct having a (Cys=Cys) double-stranded bond (DSB) within the linker.

[0088] Embodiment 59. The hairpin tCAR construct comprises a V H 1 and V H The first heavy (V H ) chain variable fragment and a second heavy (V H ) chain variable fragment, and L The first light (V12) derived from the second scFv, designated V2 and V13, L ) chain variable fragment and a second light (V L ) chain variable fragment, said first heavy (V H ) chain variable fragment and the second heavy (V H ) chain variable fragment is (GGGGS) 0~1 -(GGGGC)1-(GGGGS) 1~2 -(GGGGGP)1-(GGGGS) 2~3 -(GGGGC)1-(GGGGS) 0~1 The first light (V) from the second scFv is linked by a linker. L ) chain variable fragment and the second light (V) chain variable fragment derived from the first scFv L 59. The tCAR of embodiment 58, wherein the tCAR is linked to a .) chain variable fragment.

[0089] Embodiment 60. The hairpin tCAR construct comprises a V H 2 and V H A second heavy (V H ) chain variable fragment and the first heavy (V H ) chain variable fragment, and L 1 and V LA first light (V) fragment derived from the first scFv, designated V L ) chain variable fragment and a second light (V) chain variable fragment derived from the second scFv. L ) chain variable fragment, and the second heavy (V H ) chain variable fragment and the first heavy (V H ) chain variable fragment is (GGGGS) 0~1 -(GGGGC)1-(GGGGS) 1~2 -(GGGGGP)1-(GGGGS) 2~3 -(GGGGC)1-(GGGGS) 0~1 The first light (V) fragment from the first scFv is linked by a linker. L ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv L 59. The tCAR of embodiment 58, wherein the tCAR is linked to a .) chain variable fragment.

[0090] Embodiment 61. The hairpin tCAR construct comprises a V L 1 and V L The first light (V) derived from the first scFv, designated V L ) chain variable fragment and a second light (V L ) chain variable fragment, and H 2 and V H A first heavy (V H ) chain variable fragment and a second heavy (V L ) chain variable fragment, the first light (V) chain variable fragment derived from the first scFv L ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv L ) chain variable fragment is (GGGGS) 0~1 -(GGGGC)1-(GGGGS) 1~2 -(GGGGGP)1-(GGGGS) 2~3 -(GGGGC)1-(GGGGS) 0~1 The first heavy chain (V) from the first scFv is linked by a linker. H) chain variable fragment and the second heavy (V L 59. The tCAR of embodiment 58, wherein the tCAR is linked to a .) chain variable fragment.

[0091] Embodiment 62. The hairpin tCAR construct comprises a V L 2 and V L A second light (V) antibody derived from a second scFv, designated V L ) chain variable fragment and a first light (V L ) chain variable fragment, and H 1 and V H The first heavy (V H ) chain variable fragment and a second light (V H ) chain variable fragment, and the second light (V) chain variable fragment derived from the second scFv. L ) chain variable fragment and the first light (V) chain variable fragment derived from the first scFv L ) chain variable fragment is (GGGGS) 0~1 -(GGGGC)1-(GGGGS) 1~2 -(GGGGGP)1-(GGGGS) 2~3 -(GGGGC)1-(GGGGS) 0~1 The first heavy chain (V) from the first scFv is linked by a linker. H ) chain variable fragment and the second light (V H 59. The tCAR of embodiment 58, wherein the tCAR is linked to a .) chain variable fragment.

[0092] Embodiment 63. The tCAR described in embodiment 58, wherein the hairpin DSB tCAR construct comprises a structure selected from 11-I to 11-XXXII.

[0093] Embodiment 64. Each V H Chain and V L A tCAR according to any one of embodiments 41 to 63, wherein the chains are derived from scFvs that recognize different antigens selected from CD5, CD7, CD2, CD4, and CD3.

[0094] Embodiment 65. Each V H Chain and V L A tCAR described in embodiment 64, in which the chains are different and exhibit at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 12 to SEQ ID NO: 31.

[0095] Embodiment 66. Each V H Chain and V L A tCAR described in embodiment 64, in which the chains are different and exhibit at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 12 to SEQ ID NO: 31.

[0096] 67. Each V H Chain and V L The tCAR of embodiment 64, wherein the strands are different and are sequences selected from SEQ ID NO: 12 to SEQ ID NO: 31.

[0097] Embodiment 68. A tCAR according to any one of embodiments 35, 39, and 41 to 67, comprising at least one costimulatory domain selected from CD28 and 4-1BB.

[0098] Embodiment 69. The tCAR of embodiment 68, wherein the costimulatory domain is CD28.

[0099] Embodiment 70. A tCAR according to any one of embodiments 35 and 40 to 69, comprising a CD3 zeta signaling domain.

[0100] Embodiment 71. Each of the V H Chain and V L A tCAR according to any one of embodiments 41 to 63 and embodiments 68 to 70, wherein the chain is derived from an scFv that recognizes CD2 or an scFv that recognizes CD3.

[0101] Embodiment 72. The tCAR of embodiment 64, wherein the tCAR construct is selected from clone 5, clone 6, clone 7, clone 8, clone 13, clone 14, clone 15, and clone 16.

[0102] Embodiment 73. The tCAR of embodiment 64, wherein the tCAR construct exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO: 46.

[0103] Embodiment 74. A tandem chimeric antigen receptor (CAR) T cell (tCAR-T cell) comprising a tCAR targeting two or more T cell antigens as described in any one of embodiments 35 and embodiments 40 to 73.

[0104] Embodiment 75. The tCAR-T cell of embodiment 74, wherein the cell is deficient in one or more antigens to which the one or more CARs specifically bind.

[0105] Embodiment 76. A tCAR-T cell described in any one of embodiments 74 and 75, which is missing a subunit of the T cell receptor complex.

[0106] Embodiment 77. The tCAR-T cell of embodiment 76, wherein the subunits of the T cell receptor complex are selected from TCRα (TRAC), TCRβ, TCRδ, TCRγ, CD3ε, CD3γ, CD3δ, and CD3ζ.

[0107] Embodiment 78. The tCAR-T cell of embodiment 77, wherein the subunits of the T cell receptor complex are selected from TCRα (TRAC) and CD3ε.

[0108] Embodiment 79. The tCAR-T cell of embodiment 78, wherein the subunit of the T cell receptor complex is TRAC.

[0109] Embodiment 80. The tCAR-T cell of any one of embodiments 35 and embodiments 40 to 79, wherein the CAR-T cell further comprises a suicide gene.

[0110] Embodiment 81. A tCAR-T cell according to any one of embodiments 35 and embodiments 40 to 80, wherein endogenous T cell receptor-mediated signaling is inhibited in said CAR-T cell.

[0111] Embodiment 82. The tCAR-T cell of any one of embodiments 35 and embodiments 40 to 81, wherein the CAR-T cell does not induce alloreactivity or graft-versus-host disease.

[0112] Embodiment 83. A tCAR-T cell described in any one of embodiments 35 and embodiments 40 to 82, wherein the CAR-T cell does not induce sibling killing.

[0113] Embodiment 84. A tandem CAR-T cell having a CAR that targets CD2 and CD3, wherein the CAR-T cell is defective in a subunit of the T cell receptor complex and is defective in CD2.

[0114] Embodiment 85. The CAR-T cell of embodiment 85, wherein the CAR exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO: 44.

[0115] Embodiment 86. The CAR-T cell of embodiment 85, wherein the CAR exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO: 44.

[0116] Embodiment 87. The CAR-T cell of embodiment 85, wherein the CAR is an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO: 44.

[0117] Embodiment 88. A tandem CAR-T cell having a CAR that targets CD2 and CD7, wherein the CAR-T cell is deficient in subunits of the T cell receptor complex and is deficient in CD2 and CD7.

[0118] Embodiment 89. The CAR-T cell of embodiment 88, wherein the CAR exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 45 to SEQ ID NO: 46.

[0119] Embodiment 90. The CAR-T cell of embodiment 88, wherein the CAR exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 45 to SEQ ID NO: 46.

[0120] Embodiment 91. The CAR-T cell of embodiment 88, wherein the CAR is an amino acid sequence selected from SEQ ID NO: 45 to SEQ ID NO: 46.

[0121] Embodiment 92. A CAR-T cell comprising a chimeric antigen receptor (CAR) that targets CD7, the CAR-T cell being TRAC-deficient, CD7-deficient, and comprising a CD28 costimulatory domain and a CD3 zeta signaling domain.

[0122] Embodiment 93. The CAR-T cell of embodiment 92, wherein the CAR exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 39.

[0123] Embodiment 94. The CAR-T cell of embodiment 92, wherein the CAR exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 39.

[0124] Embodiment 95. The CAR-T cell of embodiment 92, wherein the CAR is an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 39.

[0125] A therapeutic composition comprising a population of CAR-T cells according to any one of embodiments 1 to 30 and embodiments 74 to 95, or a population of CAR-T cells comprising a CAR(s) according to any one of embodiments 31 to 73, and comprising at least one therapeutically acceptable carrier and / or adjuvant.

[0126] Embodiment 96. A method for treating cancer in a patient, comprising administering to the patient in need of cancer treatment a population of genome-edited CAR-T cells, genome-edited CAR-T cells, dual CAR-T cells, or tandem CAR-T cells described in any one of embodiments 1 to 30 and embodiments 74 to 95, or a population of CAR-T cells comprising a CAR(s) described in any one of embodiments 31 to 73.

[0127] Embodiment 97. The method of embodiment 97, wherein the cancer is a hematological malignancy.

[0128] Embodiment 98. The method of embodiment 98, wherein the hematological malignancy is a T-cell malignancy.

[0129] Embodiment 99. The method of embodiment 99, wherein the T-cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).

[0130] Embodiment 100. The method of embodiment 99, wherein the T cell malignancy is non-Hodgkin's lymphoma.

[0131] Embodiment 101. The method of embodiment 99, wherein the T-cell malignancy is T-cell chronic lymphocytic leukemia (T-CLL).

[0132] Embodiment 102. The method of embodiment 98, wherein the hematological malignancy is multiple myeloma.

[0133] Embodiment 103. The method of embodiment 98, wherein the hematological malignancy is acute myeloid leukemia (AML).

[0134] Embodiment 104. A method for generating CAR-T cells as described in any of the above embodiments or herein using Cas9-CRISPR and a gRNA selected from the gRNAs disclosed herein.

[0135] Embodiment 105. A method for generating a CAR-T cell as described in any of the above embodiments or herein using Cas9-CRISPR and a gRNA selected from the gRNAs disclosed in Table 12 and Tables 15 to 47.

[0136] Embodiment 106. A method for generating CAR-T cells according to any of the above embodiments or as described herein, using Cas9-CRISPR and a gRNA selected from the gRNAs disclosed in Table 12 and the gRNAs shown in bold in Tables 15 to 47.

[0137] Embodiment 107. A method for generating a CAR-T cell as described in any of the above embodiments or herein using Cas9-CRISPR and a gRNA selected from the gRNAs disclosed in Table 12.

[0138] M

[0139] Disclosed herein are genome-edited CAR-T cells derived from helper T cells, cytotoxic T cells, virus-specific cytotoxic T cells, memory T cells, or gamma delta (γδ) T cells and comprising one or more chimeric antigen receptors (CARs) that target one or more antigens, wherein the CAR-T cells are deficient in the one or more antigens to which the one or more CARs specifically bind.

[0140] Also provided are genome-edited CAR-T cells derived from helper T cells, cytotoxic T cells, virus-specific cytotoxic T cells, memory T cells, or gamma delta (γδ) T cells and comprising one or more chimeric antigen receptors (CARs) that target one or more antigens, where the CAR-T cells are lacking a subunit of the T cell receptor complex and the one or more antigens to which the one or more CARs specifically bind.

[0141] Also provided are CAR-T cells derived from helper T cells, cytotoxic T cells, virus-specific cytotoxic T cells, memory T cells, or gamma delta (γδ) T cells, wherein the deleted subunit of the T cell receptor complex is selected from TCR alpha, TCR beta, TCR delta, TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta.

[0142] In certain embodiments, the chimeric antigen receptor specifically binds to at least one antigen expressed on a malignant T cell.

[0143] In certain embodiments, the one or more antigens are selected from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.

[0144] In certain embodiments, the CAR-T cells further comprise a suicide gene therapy system.

[0145] In certain embodiments, endogenous T cell receptor-mediated signaling is inhibited in the CAR-T cell.

[0146] In certain embodiments, the CAR-T cells do not induce alloreactivity or graft-versus-host disease.

[0147] In certain embodiments, the CAR-T cells do not induce sibling killing.

[0148] Dual or tandem CAR-T cells are also provided.

[0149] Also provided are pharmaceutical compositions comprising a population of CAR-T cells disclosed herein and at least one therapeutically acceptable carrier and / or adjuvant.

[0150] Also provided is a method for treating a hematological malignancy comprising administering to a patient in need of treatment thereof a genome-edited CAR-T cell, a population of genome-edited CAR-T cells, wherein the population of genome-edited CAR-T cells is a mono-, dual-, or tandem CAR-T cell disclosed herein, or a pharmaceutical composition comprising the mono-, dual-, or tandem CAR-T cells disclosed herein.

[0151] In certain embodiments, the hematological malignancy is a T-cell malignancy.

[0152] In a particular embodiment, the T-cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).

[0153] In a particular embodiment, the T cell malignancy is non-Hodgkin's lymphoma.

[0154] In a particular embodiment, the T-cell malignancy is T-cell chronic lymphocytic leukemia (T-CLL).

[0155] In certain embodiments, the hematological malignancy is multiple myeloma.

[0156] In certain embodiments, the hematological malignancy is acute myeloid leukemia (AML).

[0157] CAR-T cells The present disclosure provides chimeric antigen receptor-bearing T cells (CAR-T cells), pharmaceutical compositions comprising the chimeric antigen receptor-bearing T cells (CAR-T cells), and methods for immunotherapy for the treatment of cancer, particularly hematological malignancies.

[0158] CAR-T cells are T cells that express a chimeric antigen receptor. The T cells expressing the CAR molecule may be helper T cells, cytotoxic T cells, virus-specific cytotoxic T cells, memory T cells, or gamma delta (γδ) T cells.

[0159] Chimeric antigen receptors (CARs) are recombinant fusion proteins that contain 1) an extracellular ligand-binding domain, i.e., an antigen recognition domain, 2) a transmembrane domain, and 3) a signaling domain.

[0160] The extracellular ligand binding domain is an oligopeptide or polypeptide capable of binding to a ligand. The extracellular ligand binding domain is preferably capable of interacting with a cell surface molecule, which may be a target antigen, a receptor, a peptide ligand, a protein ligand, or a target polypeptide. The extracellular ligand binding domain has an affinity constant or interaction (K) of about 0.1 pM to about 10 pM, about 0.1 pM to about 1 pM, or more preferably about 0.1 pM to about 100 nM. D ) can specifically bind to an antigen. The affinity constant or interaction (K D Methods for measuring affinity of a ligand to a target cell are well known in the art. In some cases, the extracellular ligand-binding domain is selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular disease state.

[0161] In one embodiment, the extracellular ligand binding domain is linked to a linker (e.g., GGGGS (2~6) ) connected by a light (V L ) chain variable fragment and heavy (V H The chimeric antigen receptor of the CAR-T cell may comprise a single-chain antibody fragment (scFv) comprising a single chain variable fragment, conferring specificity for either a T cell antigen or an antigen that is not specific for T cells. In one embodiment, the chimeric antigen receptor of the CAR-T cell may bind to a T cell specific antigen expressed or overexpressed on a malignant T cell, an antigen for which the CAR-T cell (e.g., genome-edited CAR-T cell) is deficient.

[0162] Non-limiting examples of antigens expressed on malignant T cells that are targeted by CARs include CD5, CD7, CD2, CD4, and CD3. In one embodiment, a CAR-T cell of the present disclosure comprises a chimeric antigen receptor having an extracellular ligand-binding domain that specifically binds to CD5.

[0163] In another embodiment, the CAR-T cell of the present disclosure comprises a chimeric antigen receptor having an extracellular ligand-binding domain that specifically binds to CD7. In other words, a CAR that specifically binds to CD7 comprises a polypeptide sequence that exhibits at least 80%, 90%, 95%, 97%, or 99% identity to an amino acid sequence selected from SEQ ID NO:20 and SEQ ID NO:21, and has the sequence (GGGGS). 3~4 The extracellular ligand-binding domains are linked to each other by a flexible linker comprising:

[0164] In another embodiment, the CAR-T cell of the present disclosure comprises a chimeric antigen receptor having an extracellular ligand-binding domain that specifically binds to CD2. In other words, a CAR that specifically binds to CD2 comprises a polypeptide sequence exhibiting at least 80%, 90%, 95%, 97%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 12, and SEQ ID NO: 13 or SEQ ID NO: 14, and SEQ ID NO: 15, and has the sequence (GGGGS). 3~4 The extracellular ligand-binding domains are linked to each other by a flexible linker comprising:

[0165] In yet another embodiment, the CAR-T cells of the present disclosure comprise a chimeric antigen receptor having an extracellular ligand-binding domain that specifically binds CD4.

[0166] In yet another embodiment, the CAR-T cell of the present disclosure comprises an extracellular ligand-binding domain of a chimeric antigen receptor that specifically binds to CD3. In other words, a CAR that specifically binds to CD3 comprises a polypeptide sequence that exhibits at least 80%, 90%, 95%, 97%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 16, and SEQ ID NO: 17 or SEQ ID NO: 18, and SEQ ID NO: 19, and has the sequence (GGGGS). 3~4 The extracellular ligand-binding domains are linked to each other by a flexible linker comprising:

[0167] Non-limiting examples of antigens expressed on the surface of leukemic cells (e.g., abnormal myeloblasts, red blood cells, or platelets) that CARs target include CD123 (IL3RA), CD371 (CLL-1, CLEC12A), CD117 (c-kit), and CD135 (FLT3), CD7, and Tim3. CARs with extracellular ligand binding domains that target these antigens may be constructed to treat leukemia, i.e., acute myeloid leukemia (AML).

[0168] Non-limiting examples of antigens expressed on the surface of multiple myeloma cells (e.g., malignant plasma cells) that CARs target include BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19. CARs may be constructed with extracellular ligand-binding domains that target these antigens to treat multiple myeloma. In another embodiment, CARs may be constructed with a portion of the APRIL protein that targets B-cell maturation antigen (BCMA) and the ligand for transmembrane activator and CAML interactor (TACI), effectively co-targeting both BCMA and TACI to treat multiple myeloma. Signal peptides direct the transport of secreted or transmembrane proteins to the cell membrane and / or cell surface, allowing for proper localization of the polypeptide. In particular, the signal peptide of the present disclosure directs an additional polypeptide, i.e., a CAR receptor, to the cell membrane, with the extracellular ligand-binding domain of the additional polypeptide being displayed on the cell surface, the transmembrane domain of the additional polypeptide spanning the cell membrane, and the signaling domain of the additional polypeptide being within the cytoplasmic portion of the cell. In one embodiment, the signal peptide is a signal peptide (SEQ ID NO:1) derived from human CD8α. In one embodiment, the signal peptide is a functional fragment of the CD8α signal peptide. A functional fragment is defined as a fragment of at least 10 amino acids of the CD8α signal peptide that directs the additional polypeptide to the cell membrane and / or cell surface. Examples of functional fragments of the human CD8α signal peptide include the amino acid sequences MALPVTALLLPLALLLHAA, MALPVTALLLP, PVTALLLPLALL, and LLLPLALLLHAARP.

[0169] Typically, the extracellular ligand-binding domain is linked to the signaling domain of a chimeric antigen receptor (CAR) by a transmembrane domain (Tm), which spans the cell membrane and anchors the CAR to the T cell surface, and links the extracellular ligand-binding domain to the signaling domain, affecting the expression of the CAR on the T cell surface.

[0170] The very distinctive feature of the transmembrane domain in the present disclosure is its ability to be expressed on the surface of immune cells and induce immune cell responses against a given target cell.The transmembrane domain may be derived from natural or synthetic sources.Alternatively, the transmembrane domain of the present disclosure may be derived from any membrane-associated or transmembrane protein.

[0171] Non-limiting examples of transmembrane polypeptides of the present disclosure are the alpha, beta or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CDSO, CD86, CD134, CD137 and CD154. Alternatively, the transmembrane domain may be synthetic and comprise predominantly hydrophobic amino acid residues (e.g., leucine and valine). In one embodiment, the transmembrane domain is derived from the T cell surface glycoprotein CD8 alpha chain type I isoform precursor (NP_001139345.1) (SEQ ID NO: 4), more preferably from CD28 (SEQ ID NO: 3). The transmembrane domain may further comprise a hinge region between the extracellular ligand binding domain and the transmembrane domain. The term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the hinge region is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. The hinge region may be derived from all or part of a natural molecule, such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3zeta, the alpha or beta chain of the T-cell receptor, CD45, CD4, CD5, CD8, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, ICOS, CD154, or all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or the hinge region may be a completely synthetic hinge sequence. In one embodiment, the hinge domain comprises a portion of human CD8α (SEQ ID NO:2), FcγRIIIα, or IgG1, to which it has at least 80%, 90%, 95%, 97%, or 99% sequence identity.

[0172] The chimeric antigen receptor (CAR) of the present disclosure comprises a signaling domain or an intracellular signaling domain of the CAR that influences intracellular signaling after the extracellular ligand binding domain binds to a target, resulting in the activation of immune cells and immune responses. In other words, the signaling domain influences the activation of at least one normal effector function in immune cells expressing the CAR. For example, the effector function of a T cell may be cytolytic activity or helper T cell activity, including the secretion of cytokines. Thus, the term "signaling domain" refers to the portion of a protein that transmits an effector function signal to allow a cell to perform a specialized function.

[0173] Examples of signaling domains for use in CARs may be the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction after antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capabilities. The signaling domains include two different classes of cytoplasmic signaling sequences, one class that initiates antigen-dependent primary activation, and the other class that acts antigen-independently to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequences may include signaling motifs known as ITAM immunoreceptor tyrosine-based activation motifs. ITAMs are distinct signaling motifs present in the cytoplasmic tails of various receptors that function as binding sites for tyrosine kinases of the syk / zap70 class. Non-limiting examples of ITAMs that can be used in the present disclosure include ITAMs derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In one embodiment, the signaling domain of the CAR may comprise a CD3 zeta signaling domain having an amino acid sequence of at least 80%, 90%, 95%, 97%, or 99% sequence identity to those ITAMs.

[0174] In addition, the CAR-T cells of the present disclosure may further comprise one or more suicide gene therapy systems. Suitable suicide gene therapy systems known in the art include, but are not limited to, some Herpes Simplex Virus Thymidine Kinase (HSVtk) / Ganciclovir (GCV), or inducible caspase 9 protein. In one embodiment, the suicide gene is chimeric CD34 / thymidine kinase.

[0175] The T cells disclosed herein may be resistant to sibling killing by being deficient in an antigen that is specifically bound by the chimeric antigen receptor. In some embodiments, the antigen of the T cell is modified such that the chimeric antigen receptor no longer specifically binds to the modified antigen. For example, the epitope of the antigen that the chimeric antigen receptor recognizes may be modified by one or more amino acid mutations (e.g., substitution or deletion), or the epitope may be deleted from the antigen. In other embodiments, expression of the antigen is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. Methods for reducing protein expression are well known in the art and include, but are not limited to, modifying or replacing a promoter operably linked to a nucleic acid sequence encoding the protein. In still other embodiments, the T cell is modified such that the antigen is not expressed, for example, by deleting or disrupting the gene encoding the antigen. In each of the above embodiments, the T cell may be deficient in one or preferably all of the antigens that the chimeric antigen receptor specifically binds to. The method for genetically modifying T cells to lack antigen is well known in the art, and non-limiting examples are described above.In exemplary embodiments, CRISPR / Cas9 gene editing can be used to modify T cells to lack antigen, for example, the antigen described below.Alternatively, TALEN can be used to edit genes.

[0176] In a variation of the above method, the cells may be transduced with a construct encoding one or more protein expression blockers (PEBL), either as an editing step or part of the editing step, or as part of the CAR transduction. For example, a construct encoding an antibody-derived single chain variable fragment specific for CD3ε may be transduced, for example, by a lentiviral vector. Once expressed, PEBL colocalizes with CD3ε in the cell and inhibits the expression of surface CD3 and TCRαβ. Thus, inhibition of surface CD3 / TCRαβ expression by PEBL is an alternative method for preparing allogeneic CAR-T cells. Furthermore, expression of PEBL and CAR may be combined in a single construct. Any of these methods may be performed using the methods disclosed herein, and PEBL may be generated to inhibit any of the gene suppression targets disclosed herein.

[0177] The above method can be adapted to insert CAR into the locus for the gene coding for antigen, cell surface protein, or secretable protein, such as cytokine. In this method, genome editing is brought about by transfection of CAR. Cells can then be activated as described herein, eliminating the separate genome editing step in certain embodiments. In theory, such steps should be carried out while cells are actively dividing. Such methods are also expected to bring about robust growth of genetically modified cells.

[0178] In certain circumstances, T cells may be selected for deficiency of the antigen to which the chimeric antigen receptor specifically binds. If certain T cells produce and present fewer surface proteins, but instead the antigen targeted by the T-CAR is deleted or non-functional, T cells may be selected for deficiency of the antigen and a population of antigen-deficient cells may be expanded for transduction of the CAR. Such cells may also be resistant to sibling killing. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0179] MonoCAR-T cells (mCAR-T) The CAR-T cells encompassed by the present disclosure are deficient in one or more antigens to which the chimeric antigen receptor specifically binds, and are therefore resistant to sibling killing. In some embodiments, the one or more antigens of the T cells are modified such that the chimeric antigen receptor no longer specifically binds to the one or more modified antigens. For example, the epitopes of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid mutations (e.g., substitutions or deletions), or the epitopes may be deleted from the antigens. In other embodiments, the expression of the one or more antigens is reduced in the T cells by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. Methods for reducing protein expression are well known in the art, including, but not limited to, modifying or replacing a promoter operably linked to a nucleic acid sequence encoding the protein. In still other embodiments, the T cells are modified to not express one or more antigens, for example, by deleting or disrupting the genes encoding the one or more antigens. In each of the above embodiments, the CAR-T cells may be deficient in one or preferably all antigens to which the chimeric antigen receptor specifically binds. Methods for genetically modifying T cells to be deficient in one or more antigens are well known in the art, and non-limiting examples are described herein. In the embodiments described in Examples 1-6, the CRISPR-Cas9 system is used to modify T cells to be deficient in one or more antigens.

[0180] CAR-T cells encompassed by the present disclosure may further lack endogenous T cell receptor (TCR) signaling as a result of deleting a portion of the T cell receptor (TCR)-CD3 complex. In various embodiments, it may be desirable to remove or suppress endogenous TCR signaling with the CAR-T cells disclosed herein. For example, when allogeneic T cells are used to generate CAR-T cells, endogenous TCR signaling in the CAR-T cells may be suppressed or removed to prevent or suppress graft-versus-host disease (GvHD). Methods for removing or suppressing endogenous TCR signaling are well known in the art and include, but are not limited to, deleting a portion of the TCR-CD3 receptor complex, such as the TCR receptor alpha chain (TRAC), the TCR receptor beta chain (TCRβ), TCRδ, TCRγ, CD3ε, CD3γ, and / or CD3δ. By deleting part of the TCR receptor complex, TCR-mediated signaling can be inhibited, which may allow the safe use of allogeneic T cells as a source of CAR-T cells without inducing life-threatening GvHD.

[0181] In addition, the CAR-T cells encompassed by the present disclosure may further comprise one or more suicide genes as described herein.

[0182] In one embodiment, the disclosure provides a T cell comprising a chimeric antigen receptor that specifically binds to CD5, where the T cell is deficient in CD5, e.g., a CD5ΔCART5 cell. In a non-limiting example, the deficient in CD5 is by (a) modifying CD5 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD5, (b) modifying the T cell such that expression of the antigen is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modifying the T cell such that CD5 is not expressed (e.g., by deletion or disruption of the gene encoding CD5). In a further embodiment, the T cell comprises a suicide gene and / or a modification such that endogenous T cell receptor (TCR)-mediated signaling is inhibited in the T cell. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene fused in frame to the extracellular and transmembrane domains of human CD34 cDNA is expressed in CD5ΔCART5 cells.

[0183] In another embodiment, the disclosure provides a T cell comprising a chimeric antigen receptor that specifically binds to CD7, where the T cell is deficient in CD7, e.g., a CD7ΔCART7 cell. In a non-limiting example, the deficient in CD7 is by (a) modifying CD7 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD7, (b) modifying the T cell such that expression of the antigen is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modifying the T cell such that CD7 is not expressed (e.g., by deletion or disruption of the gene encoding CD7). In a further embodiment, the T cell comprises a suicide gene and / or a modification such that endogenous T cell receptor (TCR)-mediated signaling is inhibited in the T cell. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene fused in frame to the extracellular and transmembrane domains of human CD34 cDNA is expressed in CD7ΔCART7 cells.

[0184] In another embodiment, the disclosure provides a T cell comprising a chimeric antigen receptor that specifically binds to CD2, where the T cell is deficient in CD2, e.g., a CD2ΔCART2 cell. In a non-limiting example, the CD2 is deficient by (a) modifying CD2 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD2, (b) modifying the T cell such that expression of the antigen is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modifying the T cell such that CD2 is not expressed (e.g., by deletion or disruption of the gene encoding CD2). In a further embodiment, the T cell comprises a suicide gene and / or a modification such that endogenous T cell receptor (TCR)-mediated signaling is inhibited in the T cell. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene fused in frame to the extracellular and transmembrane domains of human CD34 cDNA is expressed in CD2ΔCART2 cells.

[0185] In another embodiment, the disclosure provides a T cell comprising a chimeric antigen receptor that specifically binds to CD4, where the T cell is CD4-deficient, e.g., a CD4ΔCART4 cell. In a non-limiting example, the CD4 deficiency is by (a) modifying CD4 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD4, (b) modifying the T cell such that expression of the antigen is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modifying the T cell such that CD4 is not expressed (e.g., by deletion or disruption of the gene encoding CD4). In a further embodiment, the T cell comprises a suicide gene and / or a modification such that endogenous T cell receptor (TCR)-mediated signaling is inhibited in the T cell. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene fused in frame to the extracellular and transmembrane domains of human CD34 cDNA is expressed in CD4ΔCART4 cells.

[0186] In another embodiment, the disclosure provides a T cell comprising a chimeric antigen receptor that specifically binds to CD3, wherein the T cell is deficient in CD3ε, e.g., a CD3ΔCART3e cell. In a non-limiting example, the CD3 is deficient by (a) modifying CD3 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD3, (b) modifying the T cell such that expression of the antigen is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modifying the T cell such that CD3 is not expressed (e.g., by deletion or disruption of the gene encoding CD3ε). In a further embodiment, the T cell comprises a suicide gene and / or a modification such that endogenous T cell receptor (TCR)-mediated signaling is inhibited in the T cell. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene fused in frame to the extracellular and transmembrane domains of human CD34 cDNA is expressed in CD3ΔCART3ε cells.

[0187] Disclosed are embodiments of CAR amino acid sequences that can be expressed on the surface of genome-edited CAR-T cells derived from cytotoxic T cells, memory T cells, or gamma delta (γδ) T cells. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0188] Similar methods may be used to construct other monoCAR-T cells, which are listed in Table 4 below. [Table 4-1] [Table 4-2]

[0189] Dual CAR-T cells (dCAR-T) Dual CAR-T cells (dCAR-T) may be generated by cloning the protein coding sequence of a first extracellular ligand-binding domain into a lentiviral vector containing one or more costimulatory and signaling domains, and then cloning a second protein coding sequence of a second extracellular ligand-binding domain into the same lentiviral vector containing another one or more costimulatory and signaling domains, resulting in a plasmid in which two CAR constructs are expressed from the same vector.

[0190] In one embodiment, the present disclosure provides a dual chimeric antigen receptor that specifically binds to CD5 and the TCR receptor alpha chain (TRAC). [ka] (dCAR), i.e., protein coding sequences for two CARs expressed from a single lentiviral construct, are provided, where the T cells are deficient in CD5 and TRAC (e.g., CD5*TRAC-dCARTΔCD5ΔTRAC cells). In a non-limiting example, the deficiency of CD5 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD5 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD5 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD5 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD5 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD5 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cells comprise a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in-frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD5*TRAC-CARTΔCD5ΔTRAC cells.

[0191] In a second embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD7 and TCR receptor alpha chain (TRAC), where the T cell is CD7 and TRAC deficient, e.g., a CD7*TRAC-dCARTΔCD7ΔTRAC cell. In non-limiting examples, the deficiency of CD7 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD5 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD7 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD7 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD7 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*TRAC-dCARTΔCD7ΔTRAC cells.

[0192] In a third embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD2 and TCR receptor alpha chain (TRAC), wherein the T cell is deficient in CD2 and TRAC, e.g., a CD2*TRAC-dCARTΔCD2ΔTRAC cell. In non-limiting examples, the deficiency of CD2 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD2 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD2 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD7 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD2 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*TRAC-dCARTΔCD2ΔTRAC cells.

[0193] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD4 and TCR receptor alpha chain (TRAC), where the T cell is CD4 and TRAC deficient, e.g., a CD4*TRAC-dCARTΔCD4ΔTRAC cell. In non-limiting examples, the deficiency of CD4 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD4 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD4 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD7 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD4 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD4 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD4*TRAC-dCARTΔCD4ΔTRAC cells.

[0194] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD3 and TCR receptor alpha chain (TRAC), where the T cell is deficient in CD3 and TRAC, e.g., CD3*TRAC-dCARTΔCD3TRAC cells. In a non-limiting example, the deficient CD3 and TCR receptor alpha chain (TRAC) is due to (a) modifying the CD3 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD3 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD3 and TCR receptor alpha chain (TRAC) is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modifying the T cell such that CD3 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD3 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cells comprise a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in-frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD3*TRAC-dCARTΔCD3ΔTRAC cells.

[0195] In another embodiment, the disclosure provides genetically modified T cells comprising a dCAR that specifically binds to CD2 and CD3 epsilon (ε) chains, where the T cells are deficient in CD2 and CD3 epsilon, e.g., CD2*CD3ε-dCARTΔCD2ΔCD3ε cells. In non-limiting examples, the deficiency of CD2 and CD3 epsilon (ε) chains is by (a) modifying CD3 epsilon expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD2 and CD3 epsilon, (b) modifying the T cell such that expression of CD2 and CD3 epsilon is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and CD3 epsilon are not expressed (e.g., by deletion or disruption of the genes encoding CD2 and / or CD3 epsilon). In further embodiments, the T cells comprise a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*CD3ε-dCARTΔCD2ΔCD3ε cells.

[0196] In another embodiment, the disclosure provides genetically modified T cells comprising a dCAR that specifically binds to CD4 and CD3 epsilon (ε) chains, and the T cells are deficient in CD2 and CD3ε, e.g., CD4*CD3ε-dCARTΔCD4ΔCD3ε cells. In a non-limiting example, the deficiency of CD4 and CD3ε chains is by (a) modifying CD4 and CD3 epsilon expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD4 and CD3ε, (b) modifying the T cell such that expression of CD4 and CD3ε is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD4 and CD3ε are not expressed (e.g., by deletion or disruption of the genes encoding CD4 and / or CD3ε). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD4*CD3ε-dCARTΔCD4ΔCD3ε cells.

[0197] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD5 and TCR beta (β) chain, wherein the T cell is deficient in CD5 and TCRβ, e.g., a CD5*TCRβ-dCARTΔCD5ΔTCRβ cell. In a non-limiting example, the deficiency in CD5 and TCRβ chain is due to (a) modifying CD5 and TCRβ expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD5 and TCRβ, (b) modifying the T cell such that expression of CD5 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD5 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD5 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD5*TCRβ-dCARTΔCD5ΔTCRβ cells.

[0198] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD7 and TCR beta (β) chain, and the T cell is deficient in CD5 and TCR beta, e.g., a CD7*TCRβ-dCARTΔCD7ΔTCRβ cell. In a non-limiting example, the deficiency of CD7 and TCRβ chain is by (a) modifying CD7 and TCRβ expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD7 and TCRβ, (b) modifying the T cell such that expression of CD7 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD7 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*TCRβ-dCARTΔCD7ΔTCRβ cells.

[0199] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD2 and TCR beta (β) chain, wherein the T cell is deficient in CD2 and TCRβ, e.g., a CD2*TCRβ-dCARTΔCD7ΔTCRβ cell. In a non-limiting example, the deficiency in CD2 and TCRβ chain is by (a) modifying CD2 and TCRβ expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD2 and TCRβ, (b) modifying the T cell such that expression of CD2 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD2 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*TCRβ-dCARTΔCD2ΔTCRβ cells.

[0200] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds to CD4 and TCR beta (β) chain, wherein the T cell is deficient in CD4 and TCRβ, e.g., a CD4*TCRβ-dCARTΔCD4ΔTCRβ cell. In a non-limiting example, the deficiency in CD4 and TCRβ chain is due to (a) modifying CD4 and TCRβ expressed by the T cell such that the chimeric antigen receptor no longer specifically binds to the modified CD4 and TCRβ, (b) modifying the T cell such that expression of CD4 and TCRB is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD4 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD4 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD4*TCRβ-dCARTΔCD4ΔTCRβ cells.

[0201] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD7 and CD2, where the T cell is deficient in CD7 and CD2, e.g., a CD7*CD2-dCARTΔCD7ΔCD2 cell. In a non-limiting example, the deficient in CD7 and CD2 is by (a) modifying CD7 and CD2 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD7 and CD2, (b) modifying the T cell such that expression of CD7 and CD2 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and CD2 are not expressed (e.g., by deletion or disruption of the genes encoding CD7 and / or CD2). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*CD2-dCARTΔCD7ΔCD2 cells.

[0202] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD7 and CD5, wherein the T cell is deficient in CD7 and CD5, e.g., a CD7*CD5-dCARTΔCD7ΔCD5 cell. In a non-limiting example, the deficient in CD7 and CD5 is due to (a) modifying CD7 and CD5 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD7 and CD5, (b) modifying the T cell such that expression of CD7 and CD5 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and CD5 are not expressed (e.g., by deletion or disruption of the genes encoding CD7 and / or CD5). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*CD5-dCARTΔCD7ΔCD5 cells.

[0203] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD7 and CD4, wherein the T cell is deficient in CD7 and CD4 (e.g., a CD7*CD4-dCARTΔCD7ΔCD4 cell). In a non-limiting example, the deficient in CD7 and CD4 is due to (a) modifying CD7 and CD4 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD7 and CD4, (b) modifying the T cell such that expression of CD7 and CD4 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and CD4 are not expressed (e.g., by deletion or disruption of the genes encoding CD7 and / or CD4). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, a suicide gene expressed in CD7*CD4-dCARTΔCD7ΔCD4 cells encoding a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*CD4-dCARTΔCD7ΔCD4 cells.

[0204] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD2 and CD5, wherein the T cell is deficient in CD2, CD5, and TRAC, e.g., a CD2*CD5-dCARTΔCD2ΔCD5ΔTRAC cell. In a non-limiting example, the deficient CD2 and CD5 is due to (a) modifying CD2 and CD5 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD2 and CD5, (b) modifying the T cell such that expression of CD2 and CD5 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and CD5 are not expressed (e.g., by deletion or disruption of the genes encoding CD2 and / or CD5). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*CD5-dCARTΔCD2ΔCD5 cells.

[0205] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD2 and CD4, wherein the T cell is deficient in CD2, CD4, and TRAC, e.g., a CD2*CD4-dCARTΔCD2ΔCD4ΔTRAC cell. In a non-limiting example, the deficiency of CD2 and CD4 is by (a) modifying CD2 and CD4 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD2 and CD4, (b) modifying the T cell such that expression of CD2 and CD4 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and CD4 are not expressed (e.g., by deletion or disruption of the genes encoding CD2 and / or CD4). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*CD4-dCARTΔCD2ΔCD4 cells.

[0206] In another embodiment, the disclosure provides a genetically modified T cell comprising a dCAR that specifically binds CD5 and CD4, wherein the T cell is deficient in CD5 and CD4, e.g., a CD5*CD4-dCARTΔCD5ΔCD4 cell. In a non-limiting example, the deficient in CD5 and CD4 is by (a) modifying the CD5 and CD4 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD5 and CD4, (b) modifying the T cell such that expression of CD5 and CD4 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD5 and CD4 are not expressed (e.g., by deletion or disruption of the genes encoding CD5 and / or CD4). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD5*CD4-dCARTΔCD5ΔCD4 cells.

[0207] In one embodiment, the dual CAR-T cell comprises (i) a first chimeric antigen receptor (CAR) polypeptide comprising a first signal peptide, a first extracellular ligand binding domain, a first hinge region, a first transmembrane domain, one or more costimulatory domains, and a first signaling domain, and (ii) a second chimeric antigen receptor polypeptide comprising a second signaling peptide, a second extracellular ligand binding domain, a second hinge region, a second transmembrane domain, one or more costimulatory domains, and a second signaling domain, wherein the first extracellular ligand binding domain and the second extracellular ligand binding domain have affinity for different cell surface molecules, and the dual CAR-T cell has one or more gene disruptions that result in reduced expression of the cell surface molecules in the dual CAR-T cell.

[0208] In a second embodiment, the first signal peptide is the CD8α signal sequence.

[0209] In a third embodiment, the first extracellular ligand-binding domain is V H 1 and V L 1, which is a fusion protein of the variable regions of immunoglobulin heavy and light chains linked by a short linker peptide of 5 amino acids (GGGGS). In some embodiments, this linker peptide is repeated 3 or 4 times. In some embodiments, the first antigen recognition domain is V H 1-(GGGGS) 3~4 -V L 1, or V L 1-(GGGGS) 3~4 -V H You can choose from 1.

[0210] In another embodiment, the first hinge region comprises CD8α.

[0211] In another embodiment, the first transmembrane domain is CD8 or CD28.

[0212] In some embodiments, the first costimulatory domain comprises 4-1BB, CD28, or a combination of both in either order, ie, 4-1BB-CD28, or CD28-4-1BB.

[0213] In some embodiments, the first signaling domain is CD3ζ, or the CD3ζ bi-peptide, i.e., CD3ζ-CD3ζ.

[0214] In some embodiments, the second signal peptide is the CD8α signal sequence of SEQ ID NO:1.

[0215] In some embodiments, the second extracellular ligand binding domain is H 2 and V L2, which is a fusion protein of the variable regions of immunoglobulin heavy and light chains linked by a short linker peptide of 5 amino acids (GGGGS). In some embodiments, this linker peptide is repeated 3 or 4 times. In some embodiments, the second antigen recognition domain is V H 2-(GGGGS) 3~4 -V L 2, or V L 2-(GGGGS) 3~4 -V H You can choose from 2.

[0216] In another embodiment, the second hinge region comprises CD8α.

[0217] In another embodiment, the second transmembrane domain is CD8 or CD28.

[0218] In some embodiments, the second costimulatory domain comprises 4-1BB, CD28, or a combination of both in either order, ie, 4-1BB-CD28, or CD28-4-1BB.

[0219] In some embodiments, the second signaling domain is CD3ζ, or the CD3ζ bi-peptide, i.e., CD3ζ-CD3ζ.

[0220] In some embodiments, the CAR polypeptide is H 1-(GGGGS) 3~4 -V L a first extracellular ligand binding domain fusion protein of V; H 2-(GGGGS) 3~4 -V L and a second extracellular ligand binding domain fusion protein.

[0221] In some embodiments, the CAR polypeptide is L 1-(GGGGS) 3~4 -V H a first extracellular ligand binding domain fusion protein of V; L 2-(GGGGS)3~4 -V H and a second extracellular ligand binding domain fusion protein.

[0222] In some embodiments, the CAR polypeptide is H 2-(GGGGS) 3~4 -V L 2. a first extracellular ligand binding domain fusion protein, and V H 1-(GGGGS) 3~4 -V L 1 and a second extracellular ligand binding domain fusion protein.

[0223] In some embodiments, the CAR polypeptide is L 2-(GGGGS) 3~4 -V H 2. a first extracellular ligand binding domain fusion protein, and V L 1-(GGGGS) 3~4 -V H 1 and a second extracellular ligand binding domain fusion protein.

[0224] In some embodiments, the CAR polypeptide is H 1-(GGGGS) 3~4 -V L a first extracellular ligand binding domain fusion protein of V; L 2-(GGGGS) 3~4 -V H and a second extracellular ligand binding domain fusion protein.

[0225] In some embodiments, the CAR polypeptide is L 1-(GGGGS) 3~4 -V H a first extracellular ligand binding domain fusion protein of V; H 2-(GGGGS) 3~4 -V L and a second extracellular ligand binding domain fusion protein.

[0226] In some embodiments, the CAR polypeptide isH 2-(GGGGS) 3~4 -V L 2. a first extracellular ligand binding domain fusion protein, and V L 1-(GGGGS) 3~4 -V H 1 and a second extracellular ligand binding domain fusion protein.

[0227] In some embodiments, the CAR polypeptide is L 2-(GGGGS) 3~4 -V H 2. a first extracellular ligand binding domain fusion protein, and V H 1-(GGGGS) 3~4 -V L 1 and a second extracellular ligand binding domain fusion protein.

[0228] In some embodiments, the CAR polypeptide comprises at least one high efficiency cleavage site, wherein the high efficiency cleavage site is selected from P2A, T2A, E2A, and F2A.

[0229] In some embodiments, the CAR polypeptide comprises a suicide gene.

[0230] In some embodiments, the CAR polypeptide comprises a mutated cytokine receptor.

[0231] In some embodiments, the dual CAR-T cells target two antigens selected from CD5, CD7, CD2, CD4, CD3, CD33, CD123 (IL3RA), CD371 (CLL-1, CLEC12A), CD117 (c-kit), CD135 (FLT3), BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19, APRIL, and TACI.

[0232] Another example of a dual CAR is set forth in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0233] Tandem CAR-T cells (tCAR-T) Tandem CAR-T cells (tCAR-T) are T cells that have a single chimeric antigen polypeptide that contains two different extracellular ligand-binding domains capable of interacting with two different cell surface molecules, where the extracellular ligand-binding domains are linked to each other by a flexible linker and share one or more costimulatory domains, and where binding of the first or second extracellular ligand-binding domain transduces a signal across the one or more costimulatory domains and the signaling domain.

[0234] In one embodiment, the genetically modified T cells are [ka] (tCAR), one extracellular ligand binding domain specifically binds to CD5 and a second extracellular ligand binding domain binds to the TCR receptor alpha chain (TRAC), and the T cell is CD5 and TRAC deficient, e.g., CD5*TRAC-tCARTΔCD5ΔTRAC cell. In non-limiting examples, the loss of CD5 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD5 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the tCAR no longer specifically binds to the modified CD5 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD5 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD5 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD5 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in-frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD5*TRAC-tCARTΔCD5ΔTRAC cells.

[0235] In a second embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain that specifically binds CD7 and a second extracellular ligand binding domain that binds the TCR receptor alpha chain (TRAC), and the T cell is CD7 and TRAC deficient, e.g., a CD7*TRAC-tCARTΔCD7ΔTRAC cell. In non-limiting examples, the loss of CD7 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD7 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the tCAR no longer specifically binds to the modified CD7 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD7 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and TCR receptor alpha chain (TRAC) is not expressed (e.g., by deletion or disruption of genes encoding CD7 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in-frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*TRAC-tCARTΔCD7ΔTRAC cells.

[0236] In a third embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain that specifically binds CD2 and a second extracellular ligand binding domain that binds the TCR receptor alpha chain (TRAC), and the T cell is CD2 and TRAC deficient, e.g., a CD2*TRAC-tCARTΔCD2ΔTRAC cell. In non-limiting examples, the deficiency of CD2 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD2 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the tCAR no longer specifically binds to the modified CD2 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD2 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD2 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*TRAC-tCARTΔCD2ΔTRAC cells.

[0237] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), where one extracellular ligand binding domain specifically binds CD4 and a second extracellular ligand binding domain binds the TCR receptor alpha chain (TRAC), and the T cell is CD4 and TRAC deficient, e.g., a CD4*TRAC-tCARTΔCD4ΔTRAC cell. In non-limiting examples, the deficiency of CD4 and TCR receptor alpha chain (TRAC) is by (a) modifying the CD4 and TCR receptor alpha chain (TRAC) expressed by the T cell such that the tCAR no longer specifically binds to the modified CD4 and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD4 and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD4 and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD4 and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in-frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD4*TRAC-tCARTΔCD4ΔTRAC cells.

[0238] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), where one extracellular ligand binding domain specifically binds to the CD3 epsilon (ε) chain and a second extracellular ligand binding domain binds to the TCR receptor α chain (TRAC), and the T cell is deficient in CD3ε and TRAC, e.g., a CD3ε*TRAC-tCARTΔCD3εΔTRAC cell. In non-limiting examples, the loss of CD3ε and TCR receptor alpha chain (TRAC) is by (a) modifying the CD3ε and TCR receptor alpha chain (TRAC) expressed by the T cell such that the tCAR no longer specifically binds to the modified CD3ε and TCR receptor alpha chain (TRAC), (b) modifying the T cell such that expression of CD3ε and TCR receptor alpha chain (TRAC) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD3ε and TCR receptor alpha chain (TRAC) are not expressed (e.g., by deletion or disruption of genes encoding CD3ε and / or TCR receptor alpha chain (TRAC)). In further embodiments, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD3ε*TRAC-tCARTΔCD3εΔTRAC cells.

[0239] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), where one extracellular ligand binding domain specifically binds CD2 and a second extracellular ligand binding domain binds the CD3 epsilon (ε) chain, and the T cell is deficient in CD2 and CD3ε, e.g., CD2*CD3ε-tCARTΔCD2ΔCD3ε cells. In a non-limiting example, the deficient in CD2 and CD3ε is by (a) modifying CD2 and CD3ε expressed by the T cell such that the tCAR no longer specifically binds the modified CD2 and CD3ε, (b) modifying the T cell such that expression of CD2 and CD3ε is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and CD3ε are not expressed (e.g., by deletion or disruption of the genes encoding CD2 and / or CD3ε). In further embodiments, the T cells comprise a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*CD3ε-tCARTΔCD2ΔCD3ε cells.

[0240] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), where one extracellular ligand binding domain specifically binds CD4 and a second extracellular ligand binding domain binds the CD3 epsilon (ε) chain, and the T cell is deficient in CD4 and CD3ε, e.g., CD4*CD3ε-tCARTΔCD4ΔCD3ε cells. In a non-limiting example, the deficient in CD4 and CD3ε is by (a) modifying CD4 and CD3ε expressed by the T cell such that the tCAR no longer specifically binds the modified CD4 and CD3ε, (b) modifying the T cell such that expression of CD4 and CD3ε is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD4 and CD3ε are not expressed (e.g., by deletion or disruption of the genes encoding CD4 and / or CD3ε). In further embodiments, the T cells comprise a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD4*CD3ε-tCARTΔCD4ΔCD3ε cells.

[0241] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD5 and a second extracellular ligand binding domain binds a TCRβ chain, and the T cell is deficient in CD5 and TCRβ chain, e.g., CD5*TCRβ-tCARTΔCD5ΔTCRβ cells. In a non-limiting example, the deficient CD5 and TCRβ chain is due to (a) modifying CD5 and TCRβ expressed by the T cell such that the tCAR no longer specifically binds the modified CD5 and TCRβ, (b) modifying the T cell such that expression of CD5 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD5 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD5 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD5*TCRβ-tCARTΔCD5ΔTCRβ cells.

[0242] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD7 and a second extracellular ligand binding domain binds a TCRβ chain, and the T cell is deficient in CD7 and TCRβ chain, e.g., CD7*TCRβ-tCARTΔCD7ΔTCRβ cells. In a non-limiting example, the deficient CD7 and TCRβ chain is due to (a) modifying CD7 and TCRβ expressed by the T cell such that the tCAR no longer specifically binds the modified CD7 and TCRβ, (b) modifying the T cell such that expression of CD7 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD7 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*TCRβ-tCARTΔCD7ΔTCRβ cells.

[0243] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD2 and a second extracellular ligand binding domain binds a TCRβ chain, and the T cell is deficient in CD2 and TCRβ chain, e.g., CD2*TCRβ-tCARTΔCD7ΔTCRβ cells. In a non-limiting example, the deficiency in CD2 and TCRβ chain is due to (a) modifying CD2 and TCRβ expressed by the T cell such that the tCAR no longer specifically binds the modified CD2 and TCRβ, (b) modifying the T cell such that expression of CD2 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD2 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*TCRβ-tCARTΔCD2ΔTCRβ cells.

[0244] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD4 and a second extracellular ligand binding domain binds a TCRβ chain, and the T cell is deficient in CD4 and TCRβ chain, e.g., CD4*TCRβ-tCARTΔCD4ΔTCRβ cells. In a non-limiting example, the deficiency in CD4 and TCRβ chain is due to (a) modifying CD4 and TCRβ expressed by the T cell such that the tCAR no longer specifically binds the modified CD4 and TCRβ, (b) modifying the T cell such that expression of CD4 and TCRβ is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD4 and TCRβ are not expressed (e.g., by deletion or disruption of genes encoding CD4 and / or TCRβ). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD4*TCRβ-tCARTΔCD4ΔTCRβ cells.

[0245] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD7 and a second extracellular ligand binding domain binds CD2, and the T cell is deficient in CD7 and CD2, e.g., CD7*CD2-tCARTΔCD7ΔCD2 cells. In a non-limiting example, the deficient in CD7 and CD2 is due to (a) modifying CD7 and CD2 expressed by the T cell such that the tCAR no longer specifically binds the modified CD7 and CD2, (b) modifying the T cell such that expression of CD7 and CD2 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and CD2 are not expressed (e.g., by deletion or disruption of genes encoding CD7 and / or CD2). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*CD2-tCARTΔCD7ΔCD2 cells.

[0246] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD7 and a second extracellular ligand binding domain binds CD5, and the T cell is deficient in CD7 and CD5, e.g., CD7*CD5-tCARTΔCD7ΔCD5 cells. In a non-limiting example, the deficient in CD7 and CD5 is due to (a) modifying CD7 and CD5 expressed by the T cell such that the tCAR no longer specifically binds the modified CD7 and CD5, (b) modifying the T cell such that expression of CD7 and CD5 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and CD5 are not expressed (e.g., by deletion or disruption of the genes encoding CD7 and / or CD5). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*CD5-tCARTΔCD7ΔCD5 cells.

[0247] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD7 and a second extracellular ligand binding domain binds CD4, and the T cell is deficient in CD7 and CD4, e.g., CD7*CD4-tCARTΔCD7ΔCD4 cells. In a non-limiting example, the deficient in CD7 and CD4 is due to (a) modifying CD7 and CD4 expressed by the T cell such that the tCAR no longer specifically binds the modified CD7 and CD4, (b) modifying the T cell such that expression of CD7 and CD4 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD7 and CD4 are not expressed (e.g., by deletion or disruption of the genes encoding CD7 and / or CD4). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD7*CD4-tCARTΔCD7ΔCD4 cells.

[0248] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), where one extracellular ligand binding domain specifically binds CD2 and a second extracellular ligand binding domain binds CD5, and the T cell is deficient in CD2 and CD5, e.g., a CD2*CD5-tCARTΔCD2ΔCD5 cell. In a non-limiting example, the deficient in CD2 and CD5 is due to (a) modifying CD2 and CD5 expressed by the T cell such that the tCAR no longer specifically binds the modified CD2 and CD5, (b) modifying the T cell such that expression of CD2 and CD5 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and CD5 are not expressed (e.g., by deletion or disruption of the genes encoding CD2 and / or CD5). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*CD5-tCARTΔCD2ΔCD5 cells.

[0249] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), where one extracellular ligand binding domain specifically binds CD2 and a second extracellular ligand binding domain binds CD4, and the T cell is deficient in CD2 and CD4, e.g., a CD2*CD4-tCARTΔCD2ΔCD4 cell. In a non-limiting example, the deficient in CD2 and CD4 is due to (a) modifying CD2 and CD4 expressed by the T cell such that the tCAR no longer specifically binds the modified CD2 and CD4, (b) modifying the T cell such that expression of CD2 and CD4 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD2 and CD4 are not expressed (e.g., by deletion or disruption of the genes encoding CD2 and / or CD4). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene is fused in frame to the extracellular and transmembrane domains of human CD34 cDNA and expressed in CD2*CD4-tCARTΔCD2ΔCD4 cells.

[0250] In another embodiment, the genetically modified T cell comprises a tandem chimeric antigen receptor (tCAR), one extracellular ligand binding domain specifically binds CD5 and a second extracellular ligand binding domain binds CD4, and the T cell is deficient in CD5 and CD4, e.g., a CD5*CD4-tCARTΔCD5ΔCD4 cell. In a non-limiting example, the deficient in CD5 and CD4 is by (a) modifying CD5 and CD4 expressed by the T cell such that the chimeric antigen receptor no longer specifically binds the modified CD5 and CD4, (b) modifying the T cell such that expression of CD5 and CD4 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more in the T cell, or (c) modifying the T cell such that CD5 and CD4 are not expressed (e.g., by deletion or disruption of the genes encoding CD5 and / or CD4). In a further embodiment, the T cell comprises a suicide gene. In a non-limiting example, the protein coding sequence of a modified human herpes simplex virus type 1-thymidine kinase (TK) gene fused in frame to the extracellular and transmembrane domains of human CD34 cDNA is expressed in CD5*CD4-tCARTΔCD5ΔCD4 cells.

[0251] In another embodiment, the linear tandem CAR-T cell comprises a chimeric antigen receptor (CAR) polypeptide comprising a first signal peptide, a first extracellular ligand binding domain, a second extracellular ligand binding domain, a hinge region, a transmembrane domain, one or more costimulatory domains, and a signaling domain, wherein the first extracellular ligand binding antigen recognition domain and the second extracellular ligand binding antigen recognition domain have affinity for different cell surface molecules, i.e., antigens on cancer cells, e.g., malignant T cells, malignant B cells, or malignant plasma cells, and the linear tandem CAR-T cell has one or more genetic modifications, deletions, or disruptions that result in reduced expression of the cell surface molecules in the linear tandem CAR-T cell.

[0252] In another embodiment, the signal peptide is the signal peptide derived from human CD8α (SEQ ID NO:1).

[0253] In a third embodiment, the first extracellular ligand-binding domain comprises a V linked by a linker (e.g., GGGGS). H 1 and V L 1, called the weight (V H ) chain variable fragment and light (V L In some embodiments, the first antigen recognition domain comprises a single chain antibody fragment (scFv) comprising 1) a V H 1-(GGGGS) 3~4 -V L 1 or 2) V L 1-(GGGGS) 3~4 -V H You can choose from 1.

[0254] In some embodiments, the second extracellular ligand binding domain comprises a V linked by a linker (e.g., GGGGS). H 2 and V L 2 called heavy (V H ) chain variable fragment and light (V L In some embodiments, the first antigen recognition domain comprises a single chain antibody fragment (scFv) comprising 1) a V H 2-(GGGGS) 3~4 -V L 2, or 2) V L 2-(GGGGS) 3~4 -V H You can choose from 2.

[0255] In further embodiments, the first and second antigen recognition domains are linked by a short linker peptide of 5 amino acids (GGGGS), which in some embodiments is repeated 2, 3, 4, 5, or 6 times.

[0256] Linear tandem CAR constructs In one embodiment of a linear tandem CAR construct, the first extracellular ligand binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by H 1 and V L 1, called the weight (V H ) chain variable fragment and light (V L The second extracellular ligand-binding antigen recognition domain comprises a linker (e.g., GGGGS) 2~6 V connected by L 2 and V H 2 called light (V L ) chain variable fragment and heavy (V H ) chain variable fragment.

[0257] In a second embodiment of the linear tandem CAR construct, the first extracellular ligand-binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by H 2 and V L 2 called heavy (V H ) chain variable fragment and light (V L The second extracellular ligand-binding antigen recognition domain comprises a linker (e.g., GGGGS) 2~6 V connected by L 1 and V H 1 called light (V L ) chain variable fragment and heavy (V H ) chain variable fragment.

[0258] In a third embodiment of the linear tandem CAR construct, the first extracellular ligand-binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by L 1 and V HThe second extracellular ligand-binding antigen recognition domain comprises a single-chain antibody fragment (scFv) comprising a heavy (VL) chain variable fragment and a light (VH) chain variable fragment, designated 1. The second extracellular ligand-binding antigen recognition domain comprises a linker (e.g., GGGGS) 2~6 V connected by H 2 and V L 2 called light (V H ) chain variable fragment and heavy (V L ) chain variable fragment.

[0259] In a fourth embodiment of the linear tandem CAR construct, the first extracellular ligand-binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by L 2 and V H 2 called heavy (V L ) chain variable fragment and light (V H The second extracellular ligand-binding antigen recognition domain comprises a linker (e.g., GGGGS) 2~6 V connected by H 1 and V L 1 called light (V H ) chain variable fragment and heavy (V L ) chain variable fragment.

[0260] In each of the linear tandem CAR construct embodiments, the first and second extracellular ligand binding domains target a surface molecule, i.e., an antigen expressed on malignant T cells, selected from, but not limited to, BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.

[0261] Further examples of linear tandem CARs are listed in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0262] For example, the present invention provides scFv V that targets any of the antigen pairs listed in Table 6 above. H Domain and V L A linear tandem CAR construct is provided, which may incorporate domains. [Table 7-1] [Table 7-2]

[0263] Hairpin tandem CAR constructs In one embodiment of the hairpin tandem CAR construct, the first extracellular ligand binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by H 1 and V H The second extracellular ligand-binding antigen recognition domain is a single-chain antibody fragment (scFv) that contains two heavy chain variable fragments, referred to as scFv2 and scFv3. The second extracellular ligand-binding antigen recognition domain is separated by a linker (e.g., GGGGS). 2~6 V connected by L 2 and V L The antibody comprises a single-chain antibody fragment (scFv) that contains two light chain variable fragments, designated scFv1.

[0264] In a second embodiment of the hairpin tandem CAR construct, the first extracellular ligand-binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by H 2 and V HThe second extracellular ligand-binding antigen recognition domain is a single-chain antibody fragment (scFv) that contains two heavy chain variable fragments, designated scFv1 and scFv2, which are linked by a linker (e.g., GGGGS). 2~6 V connected by L 1 and V L The antibody comprises a single-chain antibody fragment (scFv) that contains two light chain variable fragments, designated scFv2.

[0265] In a third embodiment of the hairpin tandem CAR construct, the first extracellular ligand-binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by L 1 and V L The second extracellular ligand-binding antigen recognition domain is a single-chain antibody fragment (scFv) that contains two light chain variable fragments, designated scFv2 and scFv3. The second extracellular ligand-binding antigen recognition domain is separated by a linker (e.g., GGGGS). 2~6 V connected by H 2 and V H The antibody comprises a single-chain antibody fragment (scFv) that contains two heavy chain variable fragments, designated scFv1.

[0266] In a fourth embodiment of the hairpin tandem CAR construct, the first extracellular ligand-binding domain is linked to a linker (e.g., GGGGS) 2~6 V connected by L 2 and V L The second extracellular ligand-binding antigen recognition domain is a single-chain antibody fragment (scFv) that contains two light chain variable fragments, designated scFv1 and scFv2, which are linked by a linker (e.g., GGGGS). 2~6 V connected by H 1 and V H The antibody comprises a single-chain antibody fragment (scFv) that contains two heavy chain variable fragments, designated scFv2.

[0267] In each of the hairpin tandem CAR construct embodiments, the first and second extracellular ligand binding domains target a surface molecule, i.e., an antigen expressed on malignant T cells, selected from, but not limited to, BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.

[0268] Further examples of hairpin tandem CARs are listed in Table 6 above.

[0269] Further, herein, there are provided scFv Vs that target (1) CD2 and CD3, and (2) CD2 and CD7. H Domain and V L CAR constructs and CAR-T cells that may incorporate domains are provided and are described in Table 8 below. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0270] In addition, the present invention provides scFv V that targets any of the antigen pairs listed in Table 6. H Domain and V L Hairpin tandem CAR constructs are provided that may incorporate domains. [Table 9-1] [Table 9-2]

[0271] For example, in Table 10 herein, the V H Domain and V L A hairpin tandem CAR construct incorporating the domain is provided. [Table 10]

[0272] Table 11 herein also provides scFv V that target any of the antigen pairs listed in Table 6. H Domain and V L A hairpin tandem CAR construct is provided that has a double stranded bond (DSB) that may incorporate a (Cys=Cys) domain. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0273] Methods for genetically modifying CARs in dual or tandem constructs using gene editing In further embodiments, a CAR-T cell control may be generated. For example, the control CAR-T cell may contain an extracellular domain that binds to an antigen not expressed on malignant T cells. For example, if the therapeutic CAR-T cell targets a T cell antigen such as CD7 or multiple T cell antigens such as CD2 and CD3, the antigen bound by the control CAR-T cell may be CD19. Since CD19 is an antigen expressed on B cells but not on T cells, CAR-T cells with an extracellular domain adapted to bind CD19 will not bind to T cells. These CAR-T cells may be used as controls to analyze the binding efficiency and non-specific binding of CAR-T cells that target the cancer of interest and / or recognize the antigen of interest.

[0274] CARs may be further engineered as disclosed in WO2018027036A1, optionally employing variations known to those skilled in the art. Additionally, lentiviral vectors and cell lines may be obtained, and guide RNAs may be designed, validated, and synthesized, using methods well known in the art and from commercial sources, as disclosed in WO2018027036A1.

[0275] A genetically modified CAR may be introduced into T cells using a retrovirus, which allows for efficient and stable integration of the nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems (e.g., Cas3, Cas4, Cas5, Cas5e (i.e., CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (i.e., CasA), Cse2 (i.e., CasB, CasC, CasD, CasE ... ), Cse3 (i.e., CasE), Cse4 (i.e., CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 Jan;20(1):1-10.

[0276] Manipulation of PI3K signaling may be used to prevent mutant CAR-T cell differentiation due to constitutive CAR self-signaling and promote the development of long-lived memory T cells. Pharmacological inhibition of PI3K during CAR-T manufacturing and ex vivo expansion can inhibit preferential effector T cell development and restore the effector / memory ratio of CAR-T to that seen in empty vector-transduced T cells, thereby improving T cell persistence and therapeutic activity in vivo. Inhibition of p110δ PI3K can improve efficacy and memory of tumor-specific therapeutic CD8 T cells, while inhibition of p110α PI3K can improve cytokine production and antitumor efficacy. Inhibition of PI3K has been proposed because the presence of CAR on the surface of T cells can alter T cell activation and differentiation even in the absence of ligand. Constitutive self-signaling by CARs, associated with both the scFv framework and signaling domains, can lead to ectopic T cell behavior, including altered differentiation and reduced survival. This ectopic T cell behavior is important if the efficacy of CAR-T cells in patients is directly related to their in vivo longevity. The presence of the CD28 costimulatory domain increases CAR-T cell exhaustion caused by persistent CAR self-signaling, while the 4-1BB costimulatory domain is less effective. Furthermore, CD3-zeta significantly improves constitutive activation of PI3K, AKT, mTOR and glycolysis pathways, promoting the formation of short-lived effector cells over central / stem memory cells. See, for example, Zhang W.et al., “Modulation of PI3K signaling to improve CAR T cell function,” Oncotarget, 2018 Nov 9;9(88):35807-35808.

[0277] Cytokine gene deletion or suppression In addition to gene editing of TCR and cell surface proteins and antigens, genes of secretable proteins, such as cytokines and chemokines, may be edited. Such editing may be performed, for example, to suppress or prevent the onset or maintenance of cytokine release syndrome (CRS). CRS is caused by the massive and rapid release of cytokines from immune cells in response to immunotherapeutic drugs (or other immunological stimulants). Modification, disruption, or deletion of one or more cytokine or chemokine genes may be achieved using methods well known in the art, such as abolishing gene expression by changing or deleting the sequence information of the gene, removing the gene (gene silencing), etc. This can be achieved by using genetic engineering tools well known in the art, such as transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), CRISPR, etc., by transducing small hairpin RNA (shRNA), by targeting CAR to the gene sequence of the cytokine, etc.

[0278] For example, cytokines or chemokines that can be deleted from immune effector cells disclosed herein by using Cas9-CRISPR or by targeted transduction of a CAR to the gene sequence of the cytokine include the following: XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL80, CCL81, CCL82, CCL83, CCL84, CCL85, CCL86, CCL87, CCL88, CCL89, CCL90, CCL91, CCL92, CCL93, CCL94, CCL95, CCL96, CCL97, CL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CX3CL1, IL-1α, IL-1β, IL-1RA, IL-18 , IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IF N-γ, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo , Tpo, Flt-3L, SCF, M-CSF, MSP, A2M, ACKR1, ACKR2, ACKR3, ACVR1, ACVR2B, ACVRL1, ADIPOQ, AGER, AGRN, AIMP1, AREG, BMP1, BMP10, BMP15, BMP2, BMP3, BM P4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMPR2, C10orf99, C1QTNF4, C5, CCL28, CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CD109, CD36, CD4, CD40LG, CD74, CER1, C HRD, CKLF, CLCF1, CMTM1, CMTM2, CMTM3, CMTM4, CMTM5, CMTM6, CMTM7, CMTM8, CNTF, CNTFR, COPS5, CRLF1, CSF1, CSF1R, CSF2, CSF3, CSF3R, CTF1, CX3CR1,<h2 style=";text-align:left;direction:ltr">CXCL16、CXCL17、CXCR1、CXCR2、CXCR3、CXCR4、CXCR6、EBI3、EDN1、ELANE、EN G、FAM3B、FAM3C、FAM3D、FAS、FASLG、FGF2、FLT3LG、FZD4、GBP1、GDF1、GDF10 、GDF11、GDF15、GDF2、GDF3、GDF5、GDF6、GDF7、GDF9、GPI、GREM1、GREM2、GRN 、HAX1、HFE2、HMGB1、HYAL2、IFNA10、IFNA14、IFNA16、IFNA2、IFNA5、IFNA6、 IFNA8、IFNAR1、IFNAR2、IFNB1、IFNE、IFNG、IFNGR1、IFNK、IFNL1、IFNL3、IF NW1、IL10RA、IL11RA、IL12A、IL12B、IL12RB1、IL17A、IL17B、IL17C、IL17D、 IL17F、IL18BP、IL-19、IL1F10、IL1R1、IL1R2、IL1RAPL1、IL1RL1、IL1RN、IL 20RA、IL20RB、IL21、IL22、IL22RA1、IL22RA2、IL23A、IL23R、IL24、IL25、IL2 6、IL27、IL2RA、IL2RB、IL2RG、IL31、IL31RA、IL32、IL33、IL34、IL36A、IL36 B、IL36G、IL36RN、IL37、IL6R、IL6ST、INHA、INHBA、INHBB、INHBC、INHBE、IT GA4, ITGAV, ITGB1, ITGB3, KIT, KITLG, KLHL20, LFTY1, LFTY2, LIFR, LTA, LTB, LTBP1, LTBP3, LTBP4, MIF, MINOS1-, MSTN, NAMPT, NBL1, NDP, NLRP7, NO DAL, NOG, NRG1, NRP1, NRP2, OSMR, PARK7, PDPN, PF4, PF4V1, PGLYRP1, PLP2, PPBP, PXDN, SCG2, SCGB3A1, SECTM1, SLURP1, SOSTDC1, SP100, SPP1, TCAP, T GFBR1、TGFBR2、TGFBR3、THBS1、THNSL2、THPO、TIMP1、TNF、TNFRSF11、TNFRS F1A、TNFRSF9、TNFRSF10、TNFSF11、TNFSF12、TNFSF12-、TNFSF13、TNFSF13B、These include, but are not limited to, TNFSF14, TNFSF15, TNFSF18, TNFSF4, TNFSF8, TNFSF9, TRIM16, TSLP, TWSG1, TXLNA, VASN, VEGFA, VSTM1, WFIKKN1, WFIKKN2, WNT1, WNT2, WNT5A, WNT7A, and ZFP36.

[0279] The sequences of these genes are well known and available in the art.

[0280] Indications and actual standard of care for (CAR-T) therapy In some embodiments, the genome-edited immune effector cells disclosed herein and / or generated using the methods disclosed herein express one or more chimeric antigen receptors (CARs) and can be used as medicines, i.e., for the treatment of disease. In many embodiments, the cells are CAR-T cells.

[0281] The cells disclosed herein and / or produced using the methods disclosed herein can be used in immunotherapy and adoptive cell transfer to treat or manufacture medicaments for treating cancer, autoimmune diseases, infectious diseases, and other conditions.

[0282] The cancer may be a hematological malignancy or a solid tumor. Hematological malignancies include leukemia, lymphoma, multiple myeloma, and subtypes thereof. Lymphomas can be classified in various ways, often based on the basic type of malignant cells, and include Hodgkin's lymphoma (often a cancer of Reed-Sternberg cells, but sometimes originates from B cells, and all other lymphomas are non-Hodgkin's lymphomas), B-cell lymphoma, T-cell lymphoma, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, and other lymphomas as defined herein and known in the art.

[0283] B cell lymphomas include, but are not limited to, diffuse large B cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and other B cell lymphomas as defined herein and known in the art.

[0284] T-cell lymphomas include T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and other T-cell lymphomas as defined herein and known in the art.

[0285] Leukemias include acute myeloid (or myelogenous) leukemia (AML), chronic myelogenous (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (sometimes classified as lymphoma), and other leukemias as defined herein and known in the art.

[0286] Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0287] In some embodiments, the pharmaceutical agent may be used to treat cancer in a patient, particularly solid tumors such as melanoma, neuroblastoma, glioma, or cancers such as tumors of the brain, head and neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive system (e.g., ovary), upper gastrointestinal tract, pancreas, liver, renal system (e.g., kidney), bladder, prostate, and colorectum.

[0288] In another embodiment, the medicament may be used to treat cancer in a patient, particularly a hematological malignancy selected from multiple myeloma and acute myeloid leukemia (AML), and a T-cell malignancy selected from T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma, and T-cell chronic lymphocytic leukemia (T-CLL).

[0289] In some embodiments, the cells may be used to treat autoimmune diseases, such as lupus, autoimmune rheumatoid (arthritis), multiple sclerosis, transplant rejection, Crohn's disease, ulcerative colitis, dermatitis, etc. In some embodiments, the cells are chimeric autoantibody receptor T cells, or CAR-T, which present an antigen or a fragment thereof instead of an antibody fragment; in this type of adoptive cell transfer, the B cells that cause the autoimmune disease attempt to attack the genetically modified T cells and respond by killing the genetically modified T cells.

[0290] In some embodiments, the cells may be used to treat infectious diseases such as HIV and tuberculosis.

[0291] In another embodiment, the CAR-T cells of the present disclosure may undergo robust in vivo T cell expansion and may persist for extended periods of time.

[0292] In some embodiments, the treatment of a patient with the CAR-T cells of the present disclosure can be palliative, curative or preventative. The treatment can be either part of an autologous immunotherapy treatment or part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines, or cell populations used to treat a patient are derived from the patient or a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell populations used to treat a patient are derived from a donor, not from the patient.

[0293] The treatment of cancer using the CAR-T cells of the present disclosure may be combined with one or more therapies selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, radiation therapy, laser light therapy, and radiation therapy.

[0294] The administration of the CAR-T cells or population of CAR-T cells of the present disclosure is carried out by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The CAR-T cell compositions described herein, i.e., mono-CAR, dual-CAR, tandem-CAR, can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell composition of the present disclosure is preferably administered by intravenous injection.

[0295] The dose of CAR-T cells or populations of CAR-T cells was 10 per kg of body weight. 4 ~10 9 cells, preferably 10 5 ~10 6 The administration of the CAR-T cells or population of CAR-T cells may consist of administering 1 or more doses. In another embodiment, an effective amount of the CAR-T cells or population of CAR-T cells is administered in a single dose. In another embodiment, an effective amount of the cells is administered in two or more doses over a period of time. The timing of administration is within the good sense of the health care provider and is determined by the clinical condition of the patient. The CAR-T cells or population of CAR-T cells may be obtained from any source, such as a blood bank or a donor. While patient needs vary, it is within the skill of the art to calculate the optimal range of effective amounts of any CAR-T cell population(s) for a particular disease or condition. By effective amount is meant an amount that provides a therapeutic or prophylactic effect. The dosage will depend on the age, health and weight of the patient recipient, the type of concurrent treatment (if any, frequency of treatment), and the nature of the effect desired.

[0296] In another embodiment, an effective amount of the CAR-T cells or population of CAR-T cells, or a composition comprising the CAR-T cells, is administered parenterally. The administration may be intravenous. The administration of the CAR-T cells or population of CAR-T cells, or a composition comprising the CAR-T cells, may be performed by injection directly into the tumor.

[0297] In one embodiment of the present disclosure, the CAR-T cells or population of CAR-T cells are administered to the patient along with, e.g., before, simultaneously with, or following, a number of related therapeutic modalities, including, but not limited to, treatment with cytokines, or expression of cytokines in the CAR-T, that enhance T cell proliferation and persistence, and treatment including, but not limited to, treatment with IL-2, IL-7, and IL-15, or analogs thereof.

[0298] In some embodiments, the CAR-T cells or populations of CAR-T cells of the present disclosure may be used in combination with agents that inhibit immunosuppressive pathways, including, but not limited to, inhibitors of TGFβ, interleukin 10 (IL-10), adenosine, VEGF, indoleamine-2,3-dioxygenase 1 (IDO1), indoleamine-2,3-dioxygenase 2 (IDO2), tryptophan-2,3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2.

[0299] In another embodiment, the CAR-T cells or population of CAR-T cells of the present disclosure may be used in combination with a T cell checkpoint inhibitor, including but not limited to, anti-CTLA4 (ipilimumab), anti-PD1 (pembrolizumab, nivolumab, cemiplimab), anti-PDL1 (atezolizumab, avelumab, durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR.

[0300] In another embodiment, the CAR-T cells or population of CAR-T cells of the present disclosure may be used in combination with T cell agonists, including but not limited to antibodies that stimulate CD28, ICOS, OX-40, CD27, 4-1BB, CD137, GITR, and HVEM.

[0301] In another embodiment, the CAR-T cells or population of CAR-T cells of the present disclosure may be used in combination with a therapeutic oncolytic virus, including but not limited to retroviruses, picornaviruses, rhabdoviruses, paramyxoviruses, reoviruses, parvoviruses, adenoviruses, herpesviruses, and poxviruses.

[0302] In another embodiment, the CAR-T cells or population of CAR-T cells of the present disclosure may be used in combination with an immune activating therapeutic agent, such as a Toll-like receptor agonist, including but not limited to, TLR3, TLR4, TLR7, and TLR9 agonists.

[0303] In another embodiment, the CAR-T cells or population of CAR-T cells of the present disclosure may be used in combination with a stimulator of interferon genes (STING) agonist, such as cyclic GMP-AMP synthase (cGAS).

[0304] Immune effector cell aplasia, especially T-cell aplasia, is also a concern after adoptive cell transfer therapy. When the malignancy being treated is a T-cell malignancy and the CAR-T cells target a T-cell antigen, normal T cells and their progenitors expressing the antigen are depleted and the immune mechanism is compromised. Therefore, methods to manage these side effects are the guides of therapy. Such methods include the selection and retention of non-malignant T cells or progenitors, either autologous or allogeneic (optionally genetically modified to not cause or be rejected), for later expansion and reinfusion into the patient after the CAR-T cells have been depleted or inactivated. Alternatively, subtypes of TCR-bearing cells, such as normal and malignant TCR-bearing cells, can be selected and maintained. +(However, TRBC2 + cells), or alternatively, TRBC2 + (However, TRBC1 + CAR-T cells, which recognize and kill T-cell antigens (e.g., CAR-T cells but not CAR-T cells), may be used to eradicate T-cell malignancies while retaining enough normal T cells to maintain normal immune system function.

[0305] definition Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, and molecular biology. All disclosed compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0306] As used herein, the following terms have the meanings indicated. When disclosing a range of values ​​and using the notation "n1...n2" or "between n1...n2", n1 and n2 are numbers, and thus, unless otherwise specified, the notation is intended to include the numbers themselves and the range therebetween. The range may be an integer or a sequence between the numbers, including the end numbers. For example, the range "2-6 carbons" is intended to include 2, 3, 4, 5, and 6 carbons, since carbons are present in integer units. In contrast, for example, the range "1-3 μM (micromolar)" is intended to include all of 1 μM, 3 μM, and any number of significant figures between the numbers (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

[0307] The term "about" as used herein is intended to qualify the numerical value it modifies to indicate that such numerical value varies within a margin of error. In the absence of a specific margin of error, such as when listing a standard deviation for an average value set forth in a graph or table of data, the term "about" should be understood to mean a range that may include the listed numerical value, and similarly, a range that may be included by rounding up or down to that numerical value, taking into account significant digits.

[0308] The term "activation" (and its other variants) with respect to cells should generally be understood to be synonymous with "stimulating" and, as used herein, refers to the treatment of cells that results in the proliferation of a cell population. In T cells, activation is often achieved by exposure to CD2 and CD28 (and sometimes CD2 as well) agonists, typically antibodies, optionally coated onto magnetic beads or conjugated to a colloidal polymer matrix.

[0309] The term "antigen," as used herein, is a cell surface protein that is recognized by a T cell receptor or chimeric antigen receptor (i.e., is the target of a T cell receptor or chimeric antigen receptor). In the classical sense, an antigen is a substance, typically a protein, that an antibody recognizes, but a CAR may be a light (V) antibody that recognizes one or more antigen(s). L ) chain and heavy (V H The definitions overlap insofar as they include antibody-derived domains such as ribosome (RI) chains.

[0310] The term "cancer" refers to a malignant tumor or abnormal proliferation of cells in the body. Many different cancers can be characterized or identified by specific cell surface proteins or molecules. Thus, in general terms, cancer according to the present disclosure can refer to any malignant tumor treatable with immune effector cells, such as CAR-T cells described herein, which recognize and bind to cell surface proteins on cancer cells. As used herein, cancer can refer to hematological malignancies, such as multiple myeloma, T-cell malignancies, or B-cell malignancies. T-cell malignancies can include, but are not limited to, T-cell acute lymphoblastic leukemia (T-ALL) or non-Hodgkin's lymphoma. Cancer can also refer to solid tumors, including, but not limited to, cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer.

[0311] A "cell surface protein," as used herein, is a protein (or protein complex) that is expressed by a cell, at least in part, on the surface of the cell. Examples of cell surface proteins include TCR (and its subunits) and CD7.

[0312] "Chimeric antigen receptor" or "CAR", as used herein and as commonly used in the art, refers to a recombinant fusion protein that has an extracellular ligand-binding domain, a transmembrane domain, and a signaling domain that allows the cell to perform a specific function when the extracellular ligand-binding domain binds to a component present on the target cell. For example, a CAR may have an intracellular domain that activates a T cell receptor to produce a chimeric protein that exhibits specific anti-target cell immune activity, and may have antibody-based specificity for a desired antigen (e.g., a tumor antigen). First generation CARs contain an extracellular ligand-binding domain and a signaling domain (commonly CD3ζ or FcεRIγ). Second generation CARs build on the first generation CAR constructs by including an intracellular co-stimulatory domain (commonly 4-1BB or CD28). These co-stimulatory domains serve to improve the cytotoxicity and proliferation of CAR-T cells compared to first generation CARs. Third generation CARs primarily contain multiple co-stimulatory domains to improve the proliferation and persistence of CAR-T cells. Chimeric antigen receptors are distinguished from other antigen-binding factors by both their ability to bind MHC-independent antigens and their ability to transduce activation signals through their intracellular domains.

[0313] A "CAR-bearing immune effector cell" is an immune effector cell transduced with at least one CAR. A "CAR-T cell" is a T cell transduced with at least one CAR, and the CAR-T cell may be a mono-, dual-, or tandem CAR-T cell. CAR-T cells may be autologous (meaning genetically modified from the subject's own cells) or allogeneic (meaning the cells are sourced from a healthy donor), and are often genetically modified so as not to cause host-versus-graft or graft-versus-host reactions. Donor cells may also be derived from umbilical cord blood or generated from induced pluripotent stem cells.

[0314] Dual CAR-T cells (dCAR-T) may be defined as T cells that have two different chimeric antigen receptor polypeptides with affinities for different target antigens expressed within the same effector cell, with each CAR function being independent. CARs may be expressed from a single or multiple polynucleotide sequences.

[0315] A tandem CAR-T cell (tCAR-T) may be defined as a T cell bearing a single chimeric antigen polypeptide containing two different extracellular ligand-binding domains with affinities for different targets, which are linked by a peptide linker and share one or more common costimulatory domains, such that binding of either extracellular ligand-binding domain transduces a signal across the one or more common costimulatory domains and the signaling domain.

[0316] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the therapeutic condition or disorder described in this disclosure. Such administration includes the near-simultaneous co-administration of these therapeutic agents, such as by a single capsule with a fixed ratio of active ingredients, or by multiple separate capsules for each active ingredient. In addition, such administration also includes the sequential use of each type of therapeutic agent. In either case, the treatment regimen provides the beneficial effect of the drug combination in treating the condition or disorder described herein.

[0317] The term "composition" as used herein means a population of cells for immunotherapy in combination with one or more therapeutically acceptable carriers.

[0318] The term "disease", as used herein, is generally intended to be synonymous with, and used interchangeably with, the terms "disorder", "syndrome" and "condition" (as in medical condition), in that all reflect an abnormal condition in the human or animal body or one of its parts that impairs normal functioning, are typically manifested by prominent signs and symptoms, and reduce the lifespan or quality of life of a human or animal.

[0319] The term "sibicide" as used herein refers to the process that occurs when a CAR-T cell is targeted or killed by another CAR-T cell that contains the same chimeric antigen receptor as the CAR-T cell targets, because the targeted cell expresses the antigen that is specifically recognized by the chimeric antigen receptor on both cells. CAR-T cells that contain a chimeric antigen receptor that lacks the antigen that the chimeric antigen receptor specifically binds are "sibicide resistant."

[0320] The term "genome editing" as used herein means that a gene is added, deleted, or modified to be non-functional. Thus, in certain embodiments, a "gene-edited CAR-T cell" is a CAR-T cell that has been added with a gene such as a CAR that recognizes at least one antigen, and / or deleted with a gene such as the gene(s) of the antigen(s) recognized by the CAR, and / or deleted or modified with a gene such as a TCR or a subunit thereof (e.g., α-chain or β-chain) to be non-functional, or deleted or modified with a subunit of the associated CD3 signaling complex or a subunit thereof (e.g., γ-chain, δ-chain, ε-chain, or ζ-chain) to be non-functional.

[0321] As used herein, "suicide gene" refers to a nucleic acid sequence introduced into a CAR-T cell using standard methods known in the art that, when activated, results in the death of the CAR-T cell. Optionally, the suicide gene can facilitate tracking and elimination, i.e., killing, of the CAR-T cell in vivo. Activating the suicide gene to facilitate killing of the CAR-T cell can be performed using standard methods known in the art. Suicide gene systems known in the art include, but are not limited to, (a) herpes simplex virus (HSV)-tk, which converts the non-toxic prodrug ganciclovir (GCV) into GCV-triphosphate, resulting in cell death by halting DNA replication; (b) iCasp9 binding to the small molecule AP1903 allows dimerization and activation of the intrinsic apoptotic pathway; and (c) targetable surface antigens expressed on transduced T cells (e.g., CD20 and truncated EGFR), which allow efficient elimination of modified cells by complement / antibody-dependent cellular cytotoxicity (CDC / ADCC) following administration of the relevant monoclonal antibody.

[0322] For example, a "cancer cell" is a malignant T cell, a malignant B cell, or a malignant plasma cell.

[0323] A "malignant B cell" is a B cell derived from a B cell malignancy, including, but not limited to, chronic lymphocytic leukemia (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and B cell precursor acute lymphoblastic leukemia (ALL).

[0324] A "malignant T cell" is a T cell derived from a T cell malignancy.

[0325] The term "T cell malignancies" refers to a broad and highly heterogeneous group of malignancies that originate from T cell precursors, mature T cells, or natural killer cells. Non-limiting examples of T cell malignancies include T cell acute lymphoblastic leukemia / lymphoma (T-ALL), human T cell leukemia virus type 1 positive (HTLV-1+) adult T cell leukemia / lymphoma (ATL), T cell prolymphocytic leukemia (T-PLL), adult T cell lymphoma / leukemia (HTLV-1 associated), aggressive NK cell leukemia, anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic large cell lymphoma (ALCL), ALK-negative angioimmunoblastic T cell lymphoma (AITL), breast implant associated anaplastic large cell lymphoma, chronic lymphoproliferative disorder of NK cells, extranodal nasal type NK / T cell lymphoma, enteropathy type T cell lymphoma, follicular T cell lymphoma, hepatic Splenic T-cell lymphoma, indolent T-cell lymphoproliferative disorder of the GI tract, monomorphic epitheliotropic intestinal T-cell lymphoma, mycosis fungoides, nodal peripheral T-cell lymphoma with TFH phenotype, peripheral T-cell lymphoma (PTCL), NOS, primary cutaneous γδ T-cell lymphoma, primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous apical These include acro-CD8+ T-cell lymphoma, primary cutaneous CD4+ small / medium T-cell lymphoproliferative disorder [primary cutaneous anaplastic large cell lymphoma (C-ALCL), lymphocytic papulopathy], Sézary syndrome, subcutaneous panniculitis-like T-cell lymphoma, childhood systemic EBV+ T-cell lymphoma, and T-cell large granular lymphocytic leukemia (LGL).

[0326] A "healthy donor," as used herein, is a healthy donor who does not have a hematological malignancy (eg, a T-cell malignancy).

[0327] The term "therapeutically acceptable" means a material that is suitable for use in contact with the tissues of a patient without undue toxicity, irritation, or allergic response, consistent with a reasonable benefit / risk ratio, and / or effectiveness for its intended use.

[0328] The term "therapeutically effective" is intended to qualify the amount of active ingredient used to treat a disease or disorder or to affect a clinical endpoint.

[0329] As used herein, a "secretable protein" is a protein secreted by a cell that has an effect on another cell. For example, secretable proteins include cytokines, chemokines, and transcription factors.

[0330] The term "donor template" refers to the reference genomic material that cell uses as a template to repair double-strand breaks by homology-directed repair (HDR) DNA repair pathway.Donor template contains a piece of DNA (containing the gene to be expressed, i.e., CAR or marker gene) with two homology arms that flank the double-strand break site to be inserted into genome.In some embodiments, donor template can be adeno-associated virus, single-stranded DNA, or double-stranded DNA.

[0331] The term "exposing to," as used herein, is intended to be synonymous with "culturing with" in the context of bringing a composition of interest (such as, for example, an antibody) into close contact with another composition of interest (such as, for example, a cell), and for shorter periods of contact, the term "exposing to" is intended to be used in place of the term "culturing with."

[0332] The term "patient" is generally synonymous with the term "subject" and includes all mammals, including humans. EXAMPLES

[0333] The present invention is further illustrated by the following examples.

[0334] Example 1 - Methods for generating and testing genome edited CAR-T cells by inserting a CAR into the CD3e locus The following steps may be employed to obtain genome-edited CAR-T cells, in which CAR is expressed from the gene editing locus (CAR-T) disclosed herein. In this example, the generation of CD7CARTΔCD7ΔCD3ε cells is described. As one skilled in the art will appreciate, certain steps may be performed sequentially in the order described below, or in any order, possibly resulting in different efficiencies. [Table 12]

[0335] Step 1 - T cell activation (day 0). Purify T cells from the leukopheresis chamber using the Miltenyi Human PanT Isolation Kit. Resuspend in media. Count cells. Determine the number of human T cell activating CD3 / CD28 beads needed to obtain a 3:1 bead:cell ratio. Wash beads twice with T cell media. Dilute cells in hXcyte media to 1.256 cells / mL. Add human T cell activating CD3 / CD28 beads. Aliquot 4 mL of the 1.256 cells / mL solution per well into a 6-well plate. Culture cells at 37°C.

[0336] Process 2-CRISPR (2nd day). The target gene is genetically deleted and the CAR is inserted into the gene editing locus. The DNA double-strand break may be repaired using homology-directed repair, using a donor template to repair the break and insert the desired sequence into the editing locus. The targeted deletion may be performed by electroporating Cas9 mRNA and gRNA for the target(s). The donor template may be a DNA plasmid or double-stranded linear DNA that contains homology with the DNA around the double-strand break and is electroporated with Cas9 / gRNA. In addition, a viral vector such as AAV may be used as a source of donor template. However, other techniques may be used to introduce DNA double-strand breaks. These techniques include other genome editing techniques such as TALEN and meganuclease. [Table 13]

[0337] Procedure - Nucleofection using the nucleofector 4D - 4 × 10 per reaction 6 of cells. Program EO-115 - 100ul transfection volume. All supplements need to be added to Nucleofector™ Solution P3. Prepare cell culture plates by filling the appropriate number of wells with the desired volume of recommended medium (2ml for 6-well plates) and then pre-incubating / equilibrating plates in a humidified incubator at 37°C / 5% CO2. Magnetically remove beads (do this twice to ensure complete removal). Count cells and measure cell density. Centrifuge required number of cells at 90 x g for 10 minutes at room temperature. Remove supernatant completely. Resuspend in PBS (1ml) before transferring to a microcentrifuge tube and centrifuge required number of cells at 90 x g for 10 minutes at room temperature. Remove supernatant completely. Carefully resuspend cell pellet in room temperature 4D Nucleofector™ Solution P3 (4 x 10 per 100μl). 6). Add 20ug of each gRNA (gCD7 and gCD3ε) to 15ug of Cas9 in each tube. Add 100ul of cells to the Cas9 / gRNA in each tube, mix gently and then transfer everything to the Nucleocuvette™. Tap gently to remove air bubbles. Perform electroporation using the program (Human T Cell Stimulation EO-115). After the procedure is complete, carefully remove the Nucleocuvette™ Vessel from the holder using the dedicated. Resuspend the cells in pre-warmed medium. Remove medium from the desired well and add to the cuvette and gently pipette up and down 2-3 times. Transfer to well. Repeat with medium from the same well. Incubate at 37°C.

[0338] Step 3 - Transduction of T cells with AAV vectors containing HDR repair constructs. Add the recombinant AAV6 donor vector to the cell culture 2-4 h after electroporation at an MOI of 1e4-1e6.

[0339] Step 4 - Assessment of CRISPR activity and Td efficiency (day 10) 5 × 10 from each sample 5 Remove cells and analyze by flow cytometry. Wash samples with RB. Add 3ul anti-CD34 PE antibody (this detects the CAR since our construct contains human truncated CD34). Add 5ul CD3 APC and 2ul CD2 BV421. Wash. Perform flow cytometry. Cells should be CD3ε negative, CD7 negative. Harvest T cells (day 11).

[0340] Purification of CAR-T cells. CD34+ (CAR+) and TCR-negative cells may be purified in a single step using positive selection of CD34+ cells on a Miltenyi Automacs, which enriches for CAR+ cells and removes TCR+ cells (as insertion of the CAR inhibits TCR signaling).

[0341] Step 5 - Assessing in vivo CAR-T activity If in vivo imaging experiments are to be performed, inject NSG mice with tumors (5e5 MOLT3 or HH containing luciferase). (Day 7)

[0342] Use bioluminescence imaging to image the tumor burden in mice. 2 x 10 per mouse 6 of CD34+CAR-T is injected via tail vein IV or a 4-hour chromium release assay is performed on target cells (MOLT3 or HH) (day 11). Those skilled in the art will understand that some flexibility is possible in the time periods specified in Example 1.

[0343] Example 2 - Methods for generating genome-edited tandem tCAR-T cells In a variation of the procedure in Example 1, tandem CAR-T cells that recognize two antigens may be produced. In step 2, the two antigens may be deleted from the cell surface by electroporating gRNAs for the two targets and Cas9 mRNA, or may be suppressed as described above. Then, in step 3, the CAR that recognizes the two targets is transduced into the CAR-T cells. The variation of the tandem CAR-T cells is shown in the schematic diagram of Figure 2. Another example of a tCAR-T cell is shown in Table 6.

[0344] Example 3 - Genome-edited dual or tandem CAR-T cells Using the above methods, several types of genome-edited dual CAR-T cells or genome-edited tandem CAR-T cells may be generated. Figures 1 and 2 show examples of tandem CAR-T cells and dual CAR-T cells. The figures show the target antigens but not the order of scFv expression in the tandem CAR constructs. Further examples are listed in Tables 6-11 below.

[0345] Further examples of tandem CAR-T cells and dual CAR-T cells with deletion, non-deletion, or suppression of one or more surface proteins that are the target antigens of the CAR and expressed on the CAR-T cells are described herein. Usually, the examples with deletion or suppression of two or more antigens are more likely to have the advantage of higher sibling killing resistance for these CAR-T cells. It should be further noted that the order of orientation of scFv in tandem CAR is described in Tables 6-11 but is not limited thereto. For example, CD2*CD3ε encompasses tCAR with the orientation of CD2*CD3ε or CD3ε*CD2.

[0346] Further examples of mono-CAR-T cells, tandem-CAR-T cells, and dual-CAR-T cells targeting antigens expressed on multiple myeloma cells with one or more surface proteins that are not deleted, deleted, or suppressed, which are the target antigens of the CAR and are expressed on the CAR-T cells, are described herein. Typically, examples with deletion or suppression of two or more antigens are more likely to have the advantage of higher sibling killing resistance for these CAR-T cells.

[0347] Example 4 - Treatment of patient(s) with genome-edited dual CAR-T cells or genome-edited tandem CAR-T cells Patients may be treated with cells generated by the methods described above and shown in Figures 1 and 2. For example, expanded populations of dual or tandem CAR-T cells may be infused into patients.

[0348] Dual or tandem CAR-T cells target cancer cells without inducing alloreactivity. For example, CD2*CD3ε-dCARTΔCD2ΔCD3ε cells can target cancer cells (and other non-cancer cells) that bear the CD2 and CDε surface antigens.

[0349] Example 5 - Efficacy test of CD2*CD3Δ-dCARTΔCD2ΔCD3ε using a xenogeneic model of T-ALL Efficacy test of CD2*CD3Δ-dCARTΔCD2ΔCD3ε in a xenogeneic model of T-ALL: 5×10 5 Click Beetle Red luciferase (CBR)-labeled Jurkat (T-ALL-99% CD2+, 99% CD3 by FACS) cells are injected IV into NSG recipients followed by IV injection of CD2*CD3ε-dCARTΔCD2ΔCD3ε (WC5 or WC13) control cells, CD3CARTΔCD2ΔCD3ε (UCART3) control cells, CD2CARTΔCD2ΔCD3ε (UCART2) control cells, or non-targeted CD19-CARΔCD2ΔCD3ε (UCART19) control cells on day +4. In contrast to mice receiving CD19-CARΔCD2ΔCD3ε or mice injected with tumor alone, mice receiving CD2*CD3ε-dCARTΔCD2ΔCD3ε demonstrate significantly longer survival and reduced tumor burden as measured using bioluminescence imaging, as shown in FIG. 13. In future models, CD2*CD3ε-dCARTΔCD2ΔCD3ε may confer a survival advantage over CD3CARTΔCD2ΔCD3ε, CD2CARTΔCD2ΔCD3ε, and versions of this model with deletion of either CD2 (CD2CARTΔCD2ΔCD3ε) or CD3 (CD3CARTΔCD2ΔCD3ε) target cells may reduce tumor burden.

[0350] Example 6 - Genome-edited monoCAR-T cells Examples of genome-edited monoCAR-T cells targeting antigens expressed on hematological malignancies with one or more surface proteins that are not deleted, deleted, or suppressed and that are the target antigens of the CAR and are expressed on the CAR-T cells are shown below. Usually, examples with two or more antigens deleted or suppressed are more likely to have the advantage of higher sibling killing resistance for these CAR-T cells.

[0351] Example 7 - Genome edited UCART2 / 3 cells generated by pre-activation editing On day 0, cells were thawed in thawing buffer. They were then resuspended in culture medium and left to stand for 2 hours. Cells were harvested and counted. The required number of cells was centrifuged at 100 x g for 10 minutes at room temperature. The supernatant was completely removed and cells were resuspended in PBS (1 ml) before being transferred to a microcentrifuge tube and centrifuged at 100 x g for 10 minutes at room temperature. After the supernatant was completely removed, cells were resuspended in buffer P3 and counted to 5 x 10 cells per ml. 7 The count was adjusted to 0.1%. 100 μl of cell pool volume was added to the tube containing Cas9 / gRNA and mixed gently before transferring everything to a Nucleocuvette™, which was tapped gently to remove air bubbles. Electroporation was then started using the program (Human T Cell Stimulation EO-115). After this procedure, the activated cells were transferred to pre-warmed medium and then distributed in 2 mL aliquots into 12-well plates. The aliquoted samples were left undisturbed for 24 hours.

[0352] On day 1, cells were activated with T Cell TransAct™ as shown in Table 14. [Table 14]

[0353] On day 2, 1 μl of polybrene was added for each ml of media (8 mg / ml stock). The required amount of virus was added to obtain the required MOI (multiplicity of infection). Cells and virus were mixed and returned to the 37° C. incubator.

[0354] On day 3, the activated cells were washed to remove the stimuli.

[0355] On day 12, FACS analysis identified highly pure CD3 - CD2 - / CAR-T cells are shown in Figure 5 (clone 5 and clone 6), Figure 6 (clone 7 and clone 8), Figure 7 (clone 13 and clone 14), and Figure 8 (clone 15 and clone 16). 51A standard 4-hour chromium release (Cr)-labeled genome-edited Jurkat cells (ΔCD2, ΔCD3, and ΔCD2ΔCD3) was used. 51 Cr) assays were performed. These experiments demonstrated functional tumor killing effects on CD2 and CD3 targets, independent of each other (see Figures 9A, 9B, 9C, and 9D).

[0356] Example 8 - Tumor cell killing of BCMA-CAR-T cells First, regarding the in vitro efficacy of BCMA CAR-T, 51 Standard 4-hour chromium release assay using Cr-labeled MM.1S target cells ( 51 The study was performed using a Click Beetle Red (Cr) assay. A human myeloma cell line (BCMA+ / CD19-) was modified to express Click Beetle Red luciferase fused to GFP to allow in vivo tracking (MM.1S-CG). CAR-T cells were 51 Cr-labeled MM.1S-CG cells were cultured for 4 hours at a range of effector (CAR-T) to target (MM.1S-CG) ratios and released as a marker of MM.1S-CG cell death. 51 Cr was measured (Figure 10B). Efficient killing was observed at multiple effector to target (E:T) ratios. Non-transduced activated T cells and CD19-CAR-T were used as negative controls and did not induce killing of MM.1S-CG cells. In vivo efficacy was then tested by implanting (IV) 500,000 MM.1S-CG human myeloma cells into NSG mice. After 28 days, when the tumor burden had increased, mice were left untreated or implanted with 2×10 6The mice were treated with either CD19-CAR-T or BCMA CAR-T. Compared to the control, which died around day 50, all seven mice treated with BCMA CAR-T survived to nearly 150 days or longer (Figure 10C). The cause of death of one mouse in the BCMA CAR-T cohort is unknown. Flow cytometry analysis revealed that the mouse was devoid of GFP+ tumor cells. Bioluminescence imaging (BLI) over time revealed a strong decrease in signal to background levels that never increased over the duration of the experiment (Figure 10D).

[0357] Example 9 - Tumor cell killing of CS1-CAR-T cells 5×10 5 Injection of MM.1S-CG into NSG mice demonstrated the in vivo efficacy of CS1-CAR-T cells, as well as increased total body tumor burden (BLI signal 10) after 28 days. 10 photon flux), 2×10 6 100% CS1-CAR-T cells or negative control CD19-CAR-T cells. As a way to test the specificity of CS1-CAR-T cells, mice were also implanted with MM.1S-CG cells lacking CS1 (MM.1S-CGΔCS1, using Cas9 / CRISPR technology). All mice (n=10) treated with CS1-CAR-T cells survived >90 days (Figure 11B), while the median survival of CD19 control mice (n=8) was 43 days. We treated mice implanted with MM.1S-CGΔCS1 with the above CS1-CAR-T or CD19 CAR-T. The survival of the mice was similar to that of control mice (49 days), demonstrating in vivo specificity. Bioluminescence imaging (BLI) over time showed that CS1-CAR-T treated mice showed a 3-log reduction in photon flux and elimination of bone marrow tumors (Figure 11C). A subset of CS1-CAR-T mice developed extramedullary tumors that maintained CS1 expression, suggesting that antigen escape did not occur.

[0358] Example 10 - Efficacy and cell killing of tandem targeting BCMA and CS1 (tCAR) In an attempt to improve the efficacy and killing of myeloma CAR-T cells, we designed a dual-targeting CAR-T (tCAR) expressing two scFvs in tandem, targeting BCMA and CS1. As a control, we tested a tandem CAR with mono-targeting BCMA-CAR-T cells and mono-targeting CS1-CAR-T cells side-by-side. CD19-CAR-T cells were used as a negative control. We first confirmed that each scFv was expressed in the tCAR. To do this, Jurkat cells were infected with lentiviruses expressing each CAR construct as shown in Figure 12A. CAR-T cells were cultured with human recombinant BCMA protein and human recombinant CS1 protein, each labeled with a separate fluorescent fluorophore. The negative control CAR-T cells were gated (blue) and the experimental CAR-T cells were overlaid (red). As expected, Jurkat cells expressing the CD19 CAR did not bind either BCMA or CS1 protein (lower left quadrant, Figure 12B). Jurkat cells expressing the BCMA CAR protein bound to the BCMA protein (top left quadrant, FIG. 12B). Jurkat cells expressing the CS1 CAR protein bound to the CS1 protein (bottom right quadrant, FIG. 12B). Jurkat cells expressing the tandem BCMA-CS1 CAR protein bound to both recombinant proteins (top right quadrant, FIG. 12B), indicating expression of both scFvs.

[0359] Standard 4-hour chromium release ( 51 Single and tandem CAR-T cells were tested for in vitro efficacy using a BCMA-CS1 tCART assay. In these experiments, CAR-T cells were cultured at a range of effector to target cell ratios (E:T ratios). BCMA-CS1 tCART cells killed MM.1S-CG cells with similar efficacy as both single-targeted CAR-T cells. Additional experiments will optimize the dual-targeted BCMA-CS1 CAR-T cells for in vivo efficacy.

[0360] Example 11 - Off-target analysis for gRNA selection Guide RNA was designed and activity was confirmed at Washington University Genome Engineering & iPSC. Guide RNA was designed and activity was confirmed at Washington University Genome Engineering & iPSC. Complementary sequences for any gRNA can be present throughout the genome, including but not limited to the target locus. Short sequences are more likely to hybridize to off-target. Similarly, some long sequences in gRNA can have perfect match (long_0) or near match (long_1, long_2, representing a single nucleotide difference or two nucleotide difference, respectively) throughout the genome. These may also hybridize to off-target, which may result in editing of unrelated genes, reducing editing efficiency.

[0361] Off-target analysis of the selected gRNAs was performed for two exons of hCD2 (CF58 and CF59) to determine the number of sites in the human genome that are perfect matches or contain up to one or two mismatches and may include the target site. The results for exon CF58 are listed in Table 15, and the results for exon CF59 are listed in Table 16. [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2]

[0362] The gRNA sequences in Tables 15 and 16 were normalized to gRNA activity (normalized rate to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on off-target profiles: CF58.CD2.g1 (41.2%), CF58.CD2.g23 (13.2%), CF59.CD2.g20 (26.6%), CF59.CD2.g13 (66.2%), CF59.CD2.g17 (17.5%). Guide RNAs (gRNAs) with normalized NHEJ frequency of 15% or higher are good candidates for cell line and animal model generation projects.

[0363] For hCD3E, off-target analysis of the selected gRNA was performed to determine the number of sites in the human genome that are perfect matches or contain up to 1 or 2 mismatches and may contain the target site. The results of hCD3E are listed in Table 17. [Table 17]

[0364] The gRNA sequences in Table 17 were normalized to gRNA activity (normalized rate to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on off-target profiles: MS1044.CD3E.sp28 (>15%) and MS1044.CD3E.sp12 (>15%). Guide RNAs (gRNAs) with normalized NHEJ frequency of 15% or higher are good candidates for cell line and animal model generation projects.

[0365] Off-target analysis of the selected gRNAs was performed for three exons of hCD5 (exon 3, exon 4, and exon 5) to determine the number of sites in the human genome that are perfect matches or contain up to one or two mismatches and may include the target site. The results for exon 3 are listed in Table 18, the results for exon 4 are listed in Table 19, and the results for exon 5 are listed in Table 20. [Table 18-1] [Table 18-2] [Table 18-3] [Table 19] [Table 20-1] [Table 20-2] [Table 20-3]

[0366] The gRNA sequences in Tables 18, 19, and 20 were normalized to gRNA activity (normalized ratio to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on the off-target profile: exon 3: SP597.hCD5.g2 (76.5%), SP597.hCD5.g22 (36.3%), SP597.hCD5.g39 (16.0%), SP597.hCD5.g46; exon 4: SP598.hCD5.g7, SP598.hCD5.g10 (58.5%); exon 5: SP599.hCD5.g5 (51.0%), SP599.hCD5.g30, SP599.hCD5.g42, SP599.hCD5.g58 (41.0%).

[0367] For hCSF2, off-target analysis of the selected gRNA was performed to determine the number of sites in the human genome that are perfect matches or contain up to 1 or 2 mismatches and may contain the target site. The results of hCSF2 are listed in Table 21. [Table 21]

[0368] The gRNA sequences in Table 21 were normalized to gRNA activity (normalization rate to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on off-target profiles: MS1086.CSF2.sp8 (>15%) and MS1086.CSF2.sp10 (>15%).

[0369] Off-target analysis of the selected gRNAs was performed for the two exons of hCTLA4 (exon 1 and exon 2) to determine the number of sites in the human genome that are perfect matches or contain up to one or two mismatches and may include the target site. The results for exon 1 of hCTLA4 are listed in Table 22, and the results for exon 2 of hCTLA4 are listed in Table 23. [Table 22] [Table 23-1] [Table 23-2]

[0370] The gRNA sequences in Tables 22 and 23 were normalized to gRNA activity (normalization rate to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on off-target profiles: Exon 1: SP621.hCTLA4.g2 (>15%) and SP621.hCTLA4.g12 (>15%), Exon 2: SP622.hCTLA4.g2 (>15%), SP622.hCTLA4.g9 (>15%), and SP622.hCTLA4.g33 (>15%).

[0371] Off-target analysis of the selected gRNAs was performed for two exons of hPDCD1 (CF60 and CF61) to determine the number of sites in the human genome that are perfect matches or contain up to one or two mismatches and may include the target site. The results for exon CF60 are listed in Table 24, and the results for exon CF61 are listed in Table 25. [Table 24] [Table 25-1] [Table 25-2] [Table 25-3]

[0372] The gRNA sequences in Tables 24 and 25 were normalized to gRNA activity (normalization rate to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on off-target profiles: CF60.PDCD1.g12 (65.6%), CF60.PDCD1.g3 (69.2%), CF61.PDCD1.g6, CF61.PDCD1.g2 (72.7%), and CF61.PDCD1.g35 (24.0%).

[0373] Off-target analysis of the selected gRNAs was performed for two exons of hTIM3 (exon 2 and exon 3) to determine the number of sites in the human genome that are perfect matches or contain up to 1 or 2 mismatches and may include the target site. The results for exon 2 are listed in Table 26, and the results for exon 3 are listed in Table 27. [Table 26-1] [Table 26-2] [Table 26-3] [Table 27]

[0374] The gRNA sequences in Tables 26 and 27 were normalized to gRNA activity (normalization rate to NHEJ) by next generation sequencing (NGS). GFP was used as a control. After sequencing analysis, the following gRNAs were recommended based on off-target profiles: Exon 2: SP619.hTIM3.g12 (45.0%), SP619.hTIM3.g20 (60.9%), and SP619.hTIM3.g49 (45.4%), Exon 3: SP620.hTIM3.g5 (58.0%), and SP620.hTIM3.g7 (2.9%).

[0375] Those skilled in the art can appropriately modify the methods disclosed above to generate other mono-, dual-, and tandem CAR-T cells disclosed herein and confirm their activity.

[0376] Although the invention has been described in the above examples with reference to specific details of particular embodiments thereof, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention.

Claims

1. A CAR-T cell comprising one or more chimeric antigen receptors (CARs) that target one or more antigens, the CAR-T cell being deficient in a subunit of the T cell receptor complex and / or deficient in at least one or more antigens to which the one or more CARs specifically bind.

2. A CAR-T cell comprising one or more chimeric antigen receptors (CARs) that target one or more antigens, the CAR-T cell lacking one or more antigens to which the one or more CARs specifically bind.

3. The CAR-T cell of claim 1, wherein the subunit of the T cell receptor complex is selected from TCRα, TCRβ, TCRδ, TCRγ, CD3ε, CD3γ, CD3δ, and CD3ζ.

4. The CAR-T cell of any one of claims 1 to 2, wherein the chimeric antigen receptor (CAR) specifically binds to one or more antigens expressed on malignant T cells or myeloma cells.

5. The CAR-T cell of any one of claims 1 to 4, wherein the chimeric antigen receptor (CAR) exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO:

39.

6. The CAR-T cell of any one of claims 1 to 4, wherein the chimeric antigen receptor (CAR) exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO:

39.

7. The CAR-T cell of any one of claims 1 to 4, wherein the chimeric antigen receptor (CAR) is an amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO:

39.

8. The CAR-T cell of any one of claims 1 to 4, wherein the chimeric antigen receptor(s) specifically bind to one or more antigen(s) selected from BCMA, CS1, CD38, CD138, CD19, CD33, CD123, CD371, CD117, CD135, Tim-3, CD5, CD7, CD2, CD4, CD3, CD79A, CD79B, APRIL, CD56, and CD1a.

9. The CAR-T cell of any one of claims 1 to 5, wherein the chimeric antigen receptor(s) specifically binds to at least one antigen expressed on a malignant T cell.

10. The CAR-T cell of claim 9, wherein the antigen expressed on the malignant T cell is selected from CD2, CD3, CD4, CD5, CD7, TCRA, and TCRβ.

11. The CAR-T cell of any one of claims 1 to 5, wherein the chimeric antigen receptor specifically binds to at least one antigen expressed on a malignant plasma cell.

12. The CAR-T cell of claim 11, wherein the antigen expressed on malignant plasma cells is selected from BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19.

13. The CAR-T cell of any one of claims 1 to 5, wherein the chimeric antigen receptor(s) specifically binds to at least one antigen expressed on a malignant B cell.

14. The CAR-T cell of claim 13, wherein the antigen expressed on malignant B cells is selected from CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.

15. The CAR-T cell of claim 14, wherein the antigen expressed on a malignant B cell is selected from CD19 and CD20.

16. The CAR-T cell of any one of claims 1 to 15, further comprising a suicide gene.

17. The CAR-T cell of any one of claims 1 to 16, wherein endogenous T cell receptor-mediated signaling is inhibited in the CAR-T cell.

18. The CAR-T cell of any one of claims 1 to 17, which does not induce alloreactivity or graft-versus-host disease.

19. The CAR-T cell of any one of claims 1 to 18, which does not induce sibling killing.

20. The dual or tandem CAR-T cell of any one of claims 1 to 19.

21. The CAR(s) may be CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD7, CD2xCD3 ε, CD2×CD4, CD2×CD5, CD2×CD7, CD3ε×CD4, CD3ε×CD5, CD3ε×CD7, CD4×CD5, CD4×CD7, CD5×CD7, TRAC×CD2, TRAC×CD 3ε, TRAC×CD4, TRAC×CD5, TRAC×CD7, TCRβ×CD2, TCRβ×CD3ε, TCRβ×CD4, TCRβ×CD5, TCRβ×CD7, BCMA×CS1, BCMA×CD1 9, BCMA x CD38, CS1 x CD19, CD19 x CD38, APRIL x CS1, APRIL x BCMA, APRIL x CD19, APRIL x CD38, CS1 x CD38, CD79A x BCMA , CD79A×CS1, CD79A×CD19, CD79A×CD38, CD79A×CD38, CD79A×APRIL, CD79A×CD79B, CD79B×BCMA, CD79B×CS1, CD79 The CAR-T cell of claim 20, which specifically binds to two different targets selected from BxCD19, CD79BxCD38, CD79BxAPRIL, CD79BxCD79A, CD138xBCMA, CD138xCS1, CD138xCD19, CD138xCD38, CD138xAPRIL, CD138xCD79A, CD138xCD79B, CD138xBCMA, and CD138xCS1.

22. The CAR(s) may be CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD7, CD2xCD3ε, CD2xCD4, CD2 22. The CAR-T cell of claim 21, which specifically binds to two different targets selected from: TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD5, and TCRβxCD7.

23. The CAR(s) may be BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, APRILxCD38, CS1xCD38, CD79AxBCMA, CD79AxCS1, CD79AxCD19, CD79AxCD38, CD79AxCD38, CD79AxAPRIL, CD79AxCD79B, CD79BxBCMA , CD79BxCS1, CD79BxCD19, CD79BxCD38, CD79BxAPRIL, CD79BxCD79A, CD138xBCMA, CD138xCS1, CD138xCD19, CD138xCD38, CD138xAPRIL, CD138xCD79A, CD138xCD79B, CD138xBCMA, and CD138xCS1.

24. 22. The CAR-T cell of claim 21, wherein the CAR(s) specifically bind to two different targets selected from CD123xCD371, CD123xCLEC12A, CD123xCD117, CD123xFLT3, CD123xCD7, CD123xTim3, CD371xCLEC12A, CD371xCD117, CD371xFLT3, CD371xCD7, CD371xTim3, CLEC12AxCD117, CLEC12AxFLT3, CLEC12AxCD7, CLEC12AxTim3, CD117xFLT3, CD117xCD7, CD117xTim3, FLT3xCD7, FLT3xTim3, and CD7xTim3.

25. The dual CAR-T cell of any one of claims 21 to 24.

26. The tandem CAR-T cell of any one of claims 21 to 34.

27. The CAR-T cell of any one of claims 1 to 26, further comprising a suicide gene.

28. The CAR-T cell of any one of claims 1 to 26, wherein endogenous T cell receptor-mediated signaling is inhibited in the CAR-T cell.

29. The CAR-T cell of any one of claims 1 to 26, which does not induce alloreactivity or graft-versus-host disease.

30. The CAR-T cell of any one of claims 1 to 26, which does not induce sibling killing.

31. Dual or tandem chimeric antigen receptors (dCAR or tCAR) that target two or more plasma cell antigens.

32. 32. The CAR of claim 31 , wherein the plasma cell antigen(s) is selected from BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19.

33. The CAR(s) may be BCMAxCS1, BCMAxCD19, BCMAxCD38, CS1xCD19, CD19xCD38, APRILxCS1, APRILxBCMA, APRILxCD19, APRILxCD38, CS1xCD38, CD79AxBCMA, CD79AxCS1, CD79AxCD19, CD79AxCD38, CD79AxCD38, CD79AxAPRIL, CD79AxCD79B, CD79BxBCMA 33. The CAR of claim 32, which specifically binds to two different targets selected from CD138xBCMA, CD138xCS1, CD79BxCD19, CD79BxCD38, CD79BxAPRIL, CD79BxCD79A, CD138xBCMA, CD138xCS1, CD138xCD19, CD138xCD38, CD138xAPRIL, CD138xCD79A, CD138xCD79B, CD138xBCMA, and CD138xCS1.

34. The CAR described in any one of claims 31 to 33, which is a dCAR.

35. The CAR according to any one of claims 31 to 33, which is a tCAR.

36. Dual or tandem chimeric antigen receptors (dCAR or tCAR) that target two or more leukemia cell antigens.

37. 37. The CAR of claim 36, wherein the plasma cell antigen(s) is selected from CD123, CLEC12A, CD117, FLT3, CD7, and Tim3.

38. 38. The CAR of claim 37, wherein the CAR(s) specifically bind to two different targets selected from CD123xCD371, CD123xCLEC12A, CD123xCD117, CD123xFLT3, CD123xCD7, CD123xTim3, CD371xCLEC12A, CD371xCD117, CD371xFLT3, CD371xCD7, CD371xTim3, CLEC12AxCD117, CLEC12AxFLT3, CLEC12AxCD7, CLEC12AxTim3, CD117xFLT3, CD117xCD7, CD117xTim3, FLT3xCD7, FLT3xTim3, and CD7xTim3.

39. The CAR described in any one of claims 36 to 38, which is a dCAR.

40. The CAR according to any one of claims 36 to 38, which is a tCAR.

41. Tandem chimeric antigen receptors (tCARs) target two or more T cell antigens.

42. The tCAR of claim 41, wherein the T cell antigen is selected from CD5, CD7, CD2, CD4, and CD3.

43. The tCAR of claim 42, which targets a pair (i.e., two types) of antigens.

44. The antigen pairs are: CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD4, TCRβxCD7, CD2xCD3ε, CD2x 44. The tCAR of claim 43, selected from CD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD4xCD5, CD4xCD7, CD5xCD7, TRACxCD2, TRACxCD3ε, TRACxCD4, TRACxCD5, TRACxCD7, TCRβxCD2, TCRβxCD3ε, TCRβxCD4, TCRβxCD5, and TCRβxCD7.

45. 44. The tCAR of claim 43, wherein the antigen pairs are selected from CD2xCD3ε, CD2xCD4, CD2xCD5, CD2xCD7, CD3εxCD4, CD3εxCD5, CD3εxCD7, CD4xCD5, CD4xCD7, and CD5xCD7.

46. The tCAR of any one of claims 35 and 40 to 45, wherein the CAR construct is a linear tCAR construct.

47. The linear tCAR construct comprises Ｈ 1 and V Ｌ The first weight (V Ｈ ) chain variable fragment and the first light (V Ｌ ) chain variable fragment, and Ｌ 2 and V Ｈ The second light vehicle, called V2, Ｌ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment, Ｈ ) chain variable fragment and the first light (V Ｌ ) chain variable fragment is (GGGGS) ２～６ The second light (V Ｌ ) chain variable fragment and the first heavy (V Ｈ 47. The tCAR of claim 46, wherein the tCAR is linked to a nucleotide sequence variable fragment.

48. The linear tCAR construct comprises Ｈ 2 and V Ｌ The first weight (V Ｈ ) chain variable fragment and the first light (V Ｌ ) chain variable fragment, and Ｈ 1 and V Ｌ The second light vehicle, called V1, Ｌ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment, Ｈ ) chain variable fragment and the first light (V Ｌ ) chain variable fragment is (GGGGS) ２～６ The second light (V Ｌ ) chain variable fragment and the first heavy (V Ｈ 47. The tCAR of claim 46, wherein the tCAR is linked to a nucleotide sequence variable fragment.

49. The linear tCAR construct comprises Ｌ 1 and V Ｈ The first light (V Ｌ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment, and Ｈ 2 and V Ｌ The second weight (V Ｈ ) chain variable fragment and the first light (V Ｌ ) chain variable fragment, Ｌ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment is (GGGGS) ２～６ The second moiety (V Ｈ ) chain variable fragment and the first light (V Ｌ 47. The tCAR of claim 46, wherein the tCAR is linked to a nucleotide sequence variable fragment.

50. The linear tCAR construct comprises Ｌ 2 and V Ｈ The first light (V2) Ｌ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment, and Ｈ 1 and V Ｌ The second weight (V Ｈ ) chain variable fragment and the first light (V Ｌ ) chain variable fragment, Ｌ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment is (GGGGS) ２～６ The second moiety (V Ｈ ) chain variable fragment and the first light (V Ｌ 47. The tCAR of claim 46, wherein the tCAR is linked to a nucleotide sequence variable fragment.

51. The tCAR of claim 46, wherein the linear tCAR construct comprises a structure selected from 7-I to 7-XXXII.

52. The tCAR of any one of claims 35 and 40 to 45, wherein the CAR construct is a hairpin tCAR construct.

53. The hairpin tCAR construct comprises Ｈ 1 and V Ｈ The first heavy (V Ｈ ) chain variable fragment and a second heavy (V Ｈ ) chain variable fragment, and Ｌ 2 and V １ The first light (V) derived from the second scFv is designated as V2. Ｌ ) chain variable fragment and a second light (V) chain variable fragment derived from the first scFv. Ｌ ) chain variable fragment, the first heavy (V Ｈ ) chain variable fragment and the second heavy (V Ｈ ) chain variable fragment is (GGGGS) ２～６ The first light (V) from the second scFv is connected by a linker. Ｌ ) chain variable fragment and the second light (V) chain variable fragment derived from the first scFv. Ｌ 53. The tCAR of claim 52, wherein the tCAR is linked to a .) chain variable fragment.

54. The hairpin tCAR construct comprises Ｈ 2 and V Ｈ A second heavy chain (V) derived from a second scFv, designated V Ｈ ) chain variable fragment and a first heavy (V Ｈ ) chain variable fragment, and Ｌ 1 and V Ｌ A first light (V) fragment derived from the first scFv, designated V2, Ｌ ) chain variable fragment and a second light (V) chain variable fragment derived from the second scFv. Ｌ ) chain variable fragment, and the second heavy (V) chain variable fragment derived from the second scFv. Ｈ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment is (GGGGS) ２～６ The first light (V) from the first scFv is connected by a linker. Ｌ ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv. Ｌ 53. The tCAR of claim 52, wherein the tCAR is linked to a .) chain variable fragment.

55. The hairpin tCAR construct comprises Ｌ 1 and V Ｌ The first light (V) derived from the first scFv, designated V Ｌ ) chain variable fragment and a second light (V) chain variable fragment derived from a second scFv. Ｌ ) chain variable fragment, and Ｈ 2 and V Ｈ A first heavy chain (V) derived from the first scFv, designated as scFv 1 Ｈ ) chain variable fragment and a second heavy (V) chain variable fragment derived from the second scFv. Ｌ ) chain variable fragment, the first light (V) chain variable fragment derived from the first scFv Ｌ ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv. Ｌ ) chain variable fragment is (GGGGS) ２～６ The first heavy chain (V) from the first scFv is linked by a linker. Ｈ ) chain variable fragment and the second heavy (V Ｌ 53. The tCAR of claim 52, wherein the tCAR is linked to a .) chain variable fragment.

56. The hairpin tCAR construct comprises Ｌ 2 and V Ｌ A second light (V) fragment derived from a second scFv, designated V Ｌ ) chain variable fragment and a first light (V) chain variable fragment derived from a first scFv Ｌ ) chain variable fragment, and Ｈ 1 and V Ｈ A first heavy chain (V) derived from the first scFv, designated V Ｈ ) chain variable fragment and a second light (V) chain variable fragment derived from the second scFv. Ｈ a second light (V) chain variable fragment derived from the second scFv; Ｌ ) chain variable fragment and the first light (V) chain variable fragment derived from the first scFv Ｌ ) chain variable fragment is (GGGGS) ２～６ The first heavy chain (V) from the first scFv is linked by a linker. Ｈ ) chain variable fragment and the second light (V Ｈ 53. The tCAR of claim 52, wherein the tCAR is linked to a .) chain variable fragment.

57. The tCAR of claim 52, wherein the hairpin tCAR construct comprises a structure selected from 9-I to 9-XXXII.

58. The tCAR of any one of claims 35 and 40 to 45, wherein the CAR construct is a hairpin DSB tCAR construct having a (Cys=Cys) double stranded bond (DSB) in the linker.

59. The hairpin tCAR construct comprises Ｈ 1 and V Ｈ The first heavy (V Ｈ ) chain variable fragment and a second heavy (V Ｈ ) chain variable fragment, and Ｌ 2 and V １ The first light (V) derived from the second scFv is designated as V2. Ｌ ) chain variable fragment and a second light (V) chain variable fragment derived from the first scFv. Ｌ ) chain variable fragment, the first heavy (V Ｈ ) chain variable fragment and the second heavy (V Ｈ ) chain variable fragment is (GGGGS) ０～１ -(GGGG) １ - (GGGGS) １～２ -(GGGGGP) １ - (GGGGS) ２～３ -(GGGG) １ - (GGGGS) ０～１ The first light (V) from the second scFv is connected by a linker. Ｌ ) chain variable fragment and the second light (V) chain variable fragment derived from the first scFv. Ｌ 59. The tCAR of claim 58, wherein the tCAR is linked to a .) chain variable fragment.

60. The hairpin tCAR construct comprises Ｈ 2 and V Ｈ A second heavy chain (V) derived from a second scFv, designated V Ｈ ) chain variable fragment and a first heavy (V Ｈ ) chain variable fragment, and Ｌ 1 and V Ｌ A first light (V) fragment derived from the first scFv, designated V2, Ｌ ) chain variable fragment and a second light (V) chain variable fragment derived from the second scFv. Ｌ ) chain variable fragment, and the second heavy (V) chain variable fragment derived from the second scFv. Ｈ ) chain variable fragment and the first heavy (V Ｈ ) chain variable fragment is (GGGGS) ０～１ -(GGGG) １ - (GGGGS) １～２ -(GGGGGP) １ - (GGGGS) ２～３ -(GGGG) １ - (GGGGS) ０～１ The first light (V) from the first scFv is connected by a linker. Ｌ ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv. Ｌ 59. The tCAR of claim 58, wherein the tCAR is linked to a .) chain variable fragment.

61. The hairpin tCAR construct comprises Ｌ 1 and V Ｌ The first light (V) derived from the first scFv, designated V Ｌ ) chain variable fragment and a second light (V) chain variable fragment derived from a second scFv. Ｌ ) chain variable fragment, and Ｈ 2 and V Ｈ A first heavy chain (V) derived from the first scFv, designated as scFv 1 Ｈ ) chain variable fragment and a second heavy (V) chain variable fragment derived from the second scFv. Ｌ ) chain variable fragment, the first light (V) chain variable fragment derived from the first scFv Ｌ ) chain variable fragment and the second light (V) chain variable fragment derived from the second scFv. Ｌ ) chain variable fragment is (GGGGS) ０～１ -(GGGG) １ - (GGGGS) １～２ -(GGGGGP) １ - (GGGGS) ２～３ -(GGGG) １ - (GGGGS) ０～１ The first heavy chain (V) from the first scFv is linked by a linker. Ｈ ) chain variable fragment and the second heavy (V Ｌ 59. The tCAR of claim 58, wherein the tCAR is linked to a .) chain variable fragment.

62. The hairpin tCAR construct comprises Ｌ 2 and V Ｌ A second light (V) fragment derived from a second scFv, designated V Ｌ ) chain variable fragment and a first light (V) chain variable fragment derived from a first scFv Ｌ ) chain variable fragment, and Ｈ 1 and V Ｈ A first heavy chain (V) derived from the first scFv, designated V Ｈ ) chain variable fragment and a second light (V) chain variable fragment derived from the second scFv. Ｈ a second light (V) chain variable fragment derived from the second scFv; Ｌ ) chain variable fragment and the first light (V) chain variable fragment derived from the first scFv Ｌ ) chain variable fragment is (GGGGS) ０～１ -(GGGG) １ - (GGGGS) １～２ -(GGGGGP) １ - (GGGGS) ２～３ -(GGGG) １ - (GGGGS) ０～１ The first heavy chain (V) from the first scFv is linked by a linker. Ｈ ) chain variable fragment and the second light (V Ｈ 59. The tCAR of claim 58, wherein the tCAR is linked to a .) chain variable fragment.

63. The tCAR of claim 58, wherein the hairpin DSB tCAR construct comprises a structure selected from 11-I to 11-XXXII.

64. Each V Ｈ Chain and V Ｌ 64. The tCAR of any one of claims 41 to 63, wherein the chains are derived from scFvs that recognize different antigens selected from CD5, CD7, CD2, CD4, and CD3.

65. Each V Ｈ Chain and V Ｌ The tCAR of claim 64, wherein the strands are different and exhibit at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 12 to SEQ ID NO:

31.

66. Each V Ｈ Chain and V Ｌ The tCAR of claim 64, wherein the strands are different and exhibit at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 12 to SEQ ID NO:

31.

67. Each V Ｈ Chain and V Ｌ The tCAR of claim 64, wherein the strands are different and are sequences selected from SEQ ID NO: 12 to SEQ ID NO:

31.

68. The tCAR according to any one of claims 35, 39, and 41 to 67, comprising at least one costimulatory domain selected from CD28 and 4-1BB.

69. The tCAR of claim 68, wherein the costimulatory domain is CD28.

70. 70. A tCAR according to any one of claims 35 and 40 to 69, comprising a CD3ζ signaling domain.

71. Each of the above V Ｈ Chain and V Ｌ The tCAR of any one of claims 41 to 63 and claims 68 to 70, wherein the chain is derived from an scFv that recognizes CD2 or an scFv that recognizes CD3.

72. The tCAR of claim 64, wherein the tCAR construct is selected from clone 5, clone 6, clone 7, clone 8, clone 13, clone 14, clone 15, and clone 16.

73. The tCAR of claim 64, wherein the tCAR construct exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO:

46.

74. A tandem chimeric antigen receptor (CAR) T cell (tCAR-T cell) comprising a tCAR targeting two or more T cell antigens according to any one of claims 35 and 40 to 73.

75. The tCAR-T cell of claim 74, wherein the cell is deficient in one or more antigens to which the one or more CARs specifically bind.

76. The tCAR-T cell of any one of claims 74 and 75, which is deficient in a subunit of a T cell receptor complex.

77. The tCAR-T cell of claim 76, wherein the subunit of the T cell receptor complex is selected from TCR alpha (TRAC), TCR beta, TCR delta, TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta.

78. The tCAR-T cell of claim 77, wherein the subunit of the T cell receptor complex is selected from TCRα (TRAC) and CD3ε.

79. The tCAR-T cell of claim 78, wherein said subunit of said T cell receptor complex is TRAC.

80. The tCAR-T cell of any one of claims 35 and 40 to 79, wherein the CAR-T cell further comprises a suicide gene.

81. The tCAR-T cell of any one of claims 35 and 40 to 80, wherein endogenous T cell receptor-mediated signaling is inhibited within the CAR-T cell.

82. The tCAR-T cell of any one of claims 35 and 40 to 81, wherein the CAR-T cell does not induce alloreactivity or graft-versus-host disease.

83. The tCAR-T cell of any one of claims 35 and 40 to 82, wherein the CAR-T cell does not induce sibling killing.

84. A tandem CAR-T cell having a CAR targeting CD2 and CD3, wherein the CAR-T cell is deficient in a subunit of a T cell receptor complex and is deficient in CD2.

85. The CAR-T cell of claim 85, wherein the CAR exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO:

44.

86. The CAR-T cell of claim 85, wherein the CAR exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO:

44.

87. The CAR-T cell of claim 85, wherein the CAR is an amino acid sequence selected from SEQ ID NO: 41 to SEQ ID NO:

44.

88. A tandem CAR-T cell having a CAR targeting CD2 and CD7, wherein the CAR-T cell is deficient in a subunit of a T cell receptor complex and is deficient in CD2 and CD7.

89. The CAR-T cell of claim 88, wherein the CAR exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 45 to SEQ ID NO:

46.

90. The CAR-T cell of claim 88, wherein the CAR exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 45 to SEQ ID NO:

46.

91. The CAR-T cell of claim 88, wherein the CAR is an amino acid sequence selected from SEQ ID NO: 45 to SEQ ID NO:

46.

92. A CAR-T cell comprising a chimeric antigen receptor (CAR) that targets CD7, the CAR-T cell being TRAC-deficient, CD7-deficient, and comprising a CD28 costimulatory domain and a CD3ζ signaling domain.

93. The CAR-T cell of claim 92, wherein the CAR exhibits at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO:

39.

94. The CAR-T cell of claim 92, wherein the CAR exhibits at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO:

39.

95. The CAR-T cell of claim 92, wherein the CAR is an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO:

39.

96. A therapeutic composition comprising a population of CAR-T cells according to any one of claims 1 to 30 and claims 74 to 95, or a population of CAR-T cells comprising a CAR(s) according to any one of claims 31 to 73, and comprising at least one therapeutically acceptable carrier and / or adjuvant.

97. 10. A method for treating cancer in a patient, comprising administering to a patient in need of cancer treatment a population of genome edited CAR-T cells, genome edited CAR-T cells, dual CAR-T cells, or tandem CAR-T cells of any one of claims 1 to 30 and claims 74 to 95, or a population of CAR-T cells comprising a CAR(s) of any one of claims 31 to 73.

98. 98. The method of claim 97, wherein the cancer is a hematological malignancy.

99. 99. The method of claim 98, wherein the hematological malignancy is a T-cell malignancy.

100. 100. The method of claim 99, wherein the T-cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).

101. 100. The method of claim 99, wherein the T cell malignancy is non-Hodgkin's lymphoma.

102. 100. The method of claim 99, wherein the T-cell malignancy is T-cell chronic lymphocytic leukemia (T-CLL).

103. 99. The method of claim 98, wherein the hematological malignancy is multiple myeloma.

104. 99. The method of claim 98, wherein the hematological malignancy is acute myeloid leukemia (AML).

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