Immunosuppression resistant t cells for post-transplant disorders

Abstract

The present disclosure provides engineered T cells with reduced expression or knock out of an immunophilin, and which express a chimeric antigen receptor (CAR) and / or comprise one or more modifications that disrupt MHC class I and / or MHC class II alleles and increase expression of a tolerogenic factor. The present disclosure also provides methods of treating a subject having or suspected of having a post-transplant disorder by administering T cells with reduced expression or knock out of an immunophilin. Also disclosed are pharmaceutical compositions comprising T cells with reduced expression of an immunophilin, for use in treating a subject having or suspected of having a post-transplant disorder.

Description

IMMUNUNO SUPPRESSION RESISTANT T CELLS FOR POST-TRANSPLANT DISORDERSBACKGROUND

[0001] Solid organ transplantation has the potential to save the lives of patients experiencing organ failure and to improve the quality of life of such patients. Although solid organ transplantation has been a mainstream treatment option since the 1980s, challenges remain for allograft survival.

[0002] The risk for developing lymphoproliferative disease is increased in immunocompromised patients and those that are on long-term immunosuppression. The latter include solid organ transplant recipients and their risk for post-transplant lymphoproliferative disorder (PTLD) has been shown to correlate with the level of immunosuppression.

[0003] Antibody-mediated rejection (AMR) is a primary driver of chronic rejection and can jeopardize all types of solid allografts and islets. As such, AMR is a roadblock for long-term transplant survival. Development of AMR occurs in transplant recipients across all solid organs and islets and despite use of effective systemic immunosuppression.

[0004] There is no effective approved AMR treatment, and thus AMR remains an unmet medical need. Rates of organ transplant increased 9.4% from 2022 to 2023, with more than 43,000 organs transplanted in 2023 and concomitant increases in AMR. There is an urgent need to develop therapies for such patients.SUMMARY

[0005] In patients that have received a transplant, long-term immunosuppression can lead to subsequent challenges, such as an increased risk for developing post-transplant lymphoproliferative disorder (PTLD) and antibody -mediated rejection (AMR), which is a primary driver of chronic rejection and can jeopardize all types of transplants. The standard of care for transplant patients with PTLD has traditionally involved reduction of immunosuppression and rituximab-based chemotherapy, but more recently, small case series on CAR T cell therapy have been reported. Although CAR T cell therapy has shown remarkable efficacy in non-transplant patients with relapsed or refractory diffuse large B-celllymphoma (DLBCL), its use in transplant patients is jeopardized by immunosuppression. Off-the-shelf CAR-T cells and other therapeutic cells can offer advantages over autologous cell-based strategies, including ease of manufacturing, quality control, and avoidance of malignant contamination and T cell dysfunction. However, in immunosuppressed patients, after a CAR T cell infusion, a balancing act is necessary to keep the level of immunosuppression as low as possible to enable CAR T cell killing of a tumor, while still protecting an allograft from rejection. Therefore, CAR T cells resistant to immunosuppression might offer advantages for patients that depend on immunosuppression to protect their transplanted organ. In addition, an allogeneic cell product would further allow the use of healthy donor T cells not taken from an immunosuppressed cancer patient.

[0006] The present disclosure provides engineered T cells. In some embodiments, an engineered T cell comprises one or more modifications that reduce expression of (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell. In some embodiments, an engineered T cell comprises one or more modifications that increase expression of a tolerogenic factor relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell. In some embodiments, an engineered T cell comprises modifications to express a chimeric antigen receptor (CAR).

[0007] In some embodiments, an engineered T cell comprises one or more modifications that reduce expression of FKBP12 relative to a comparable T-cell which does not comprises the modification(s) or relative to a wild-type T cell. In some embodiments, an engineered T cell comprises modifications that (a) reduce expression of FKBP12, (b) reduce expression of: (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules, (c) increase expression of a tolerogenic factor, and (d) express a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modifications or relative to a wild-type T cell.

[0008] In some embodiments, an engineered T cell comprises one or more modifications that reduce expression of cyclophilin A relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell. In some embodiments, an engineered T cell comprises modifications that (a) reduce expression of cyclophilin A, (b) reduce expression of (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules, (c) increase expression of a tolerogenic factor, and (d) express a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modifications or relative to a wild-type T cell.

[0009] The present disclosure provides pharmaceutical compositions. In some embodiments, a pharmaceutical composition comprises a population of engineered T cells. In some embodiments, a pharmaceutical composition comprises a population of engineered T cells that comprise reduced expression of (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules relative to comparable T cells or relative to wild-type T cells. In some embodiments, a pharmaceutical composition comprises a population of engineered T cells that comprise increased expression of a tolerogenic factor relative to comparable T cells or relative to wildtype T cells. In some embodiments, a pharmaceutical composition comprises engineered T cells that express a chimeric antigen receptor (CAR).

[0010] In some embodiments, a pharmaceutical composition comprises a population of engineered T cells that comprise reduced expression of FKBP12 relative to comparable T cells or relative to wild-type T cells. In some embodiments, a pharmaceutical composition comprises a population of engineered T cells that comprise (a) reduced expression of FKBP12, (b) reduced expression of (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules, (c) increased expression of a tolerogenic factor, and (d) expression a chimeric antigen receptor(CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell or relative to a wild-type T cell.

[0011] In some embodiments, a pharmaceutical composition comprises a population of engineered T cells that comprise reduced expression of cyclophilin A relative to comparable T cells or relative to wild-type T cells. In some embodiments, a pharmaceutical composition comprises a population of engineered T cells that comprise (a) reduced expression of cyclophilin A, (b) reduced expression of: (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules, (c) increased expression of a tolerogenic factor, and (d) expression a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell or a wild-type T cell.

[0012] The present disclosure also provides methods of making populations of engineered T cells. In some embodiments, a method of making engineered T cells comprises expressing a chimeric antigen receptor (CAR) in the population of T cells.

[0013] In some embodiments, a method of making engineered T cells comprises reducing expression of FKBP12 in a population of T cells relative to comparable T cells or wild-type T cells. In some embodiments, a method of making a population of engineered T cells comprises (a) reducing expression of FKBP12 in a population of T cells relative to comparable T cells or wild-type T cells, and (b) expressing a chimeric antigen receptor (CAR) in the population of T cells, thereby making a population of engineered T cells.

[0014] In some embodiments, a method of making a population of engineered T cells comprises reducing expression of cyclophilin A in a population of T cells relative to comparable T cells or wild-type T cells. In some embodiments, a method of making a population of engineered T cells comprises (a) reducing expression of cyclophilin A in a population of T cells relative to comparable T cells or wild-type T cells, and (b) expressing a chimeric antigen receptor (CAR) in the population of T cells, thereby making a population of engineered T cells.

[0015] The present disclosure provides methods comprising administering to a subject a composition comprising a population of engineered T cells. In some embodiments, a method comprises administering to a subject a composition comprising a population ofengineered T cells that comprise one or more modifications that express a chimeric antigen receptor (CAR).

[0016] In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells that comprise one or more modifications that reduce expression of FKBP12 relative to comparable T cells that do not comprise the modification(s) or relative to wild-type T cells. In some embodiments, a method comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that: (a) reduce expression of FKBP12 relative to comparable T cells that do not comprise the modification(s) or relative to wild-type T cells, and (b) express a chimeric antigen receptor (CAR).

[0017] In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells comprising one or more modifications that reduce expression of cyclophilin A relative to comparable T cells that do not comprise the modification(s) or relative to wild-type T cells. In some embodiments, a method comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that: (a) reduce expression of cyclophilin A relative to comparable T cells that do not comprise the modification(s) or relative to wild-type T cells, and (b) express a chimeric antigen receptor (CAR).

[0018] The present disclosure provides methods of treatment or prevention. In some embodiments, a method of the present disclosure comprises treating or preventing a posttransplant disorder. In some embodiments, a method of treatment or prevention comprises treating or preventing an autoimmune disease or disorder. In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells. In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that reduce expression of FKBP12 relative to comparable T cells that do not comprise the modification(s) or relative to wild-type T cells. In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that reduce expression of cyclophilin A relative to comparable T cells that do not comprise themodification(s) or relative to wild-type T cells. In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that express a chimeric antigen receptor (CAR). In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that (a) reduce expression of FKBP12 relative to comparable T cells that do not comprise the modification(s) or wild-type T cells, or (b) reduce expression of cyclophilin A relative to comparable T cells that do not comprise the modification(s) or wildtype T cells; and (c) express a chimeric antigen receptor (CAR).

[0019] In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that reduce expression of beta-2-microglobulin (B2M), Class II Major Histocompatibility Complex Transactivator (CIITA), and T cell receptor alpha constant (TRAC) relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell. In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that increase expression of a tolerogenic factor, wherein the increased expression is relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell.

[0020] In some embodiments, a method of the present disclosure comprises administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that: (a) reduce expression of FKBP12 relative to comparable T cells that do not comprise the modification(s) or wild-type T cells, or (b) reduce expression of cyclophilin A relative to comparable T cells that do not comprise the modification(s) or wild-type T cells; and (c) express a chimeric antigen receptor (CAR); (d) reduce expression of beta-2-microglobulin (B2M), Class II Major Histocompatibility Complex Transactivator (CIITA), and T cell receptor alpha constant (TRAC) relative to a comparable T cell that does not comprise the modification(s) or wildtype T cell; and (e) increase expression of a tolerogenic factor, wherein the increased expression is relative to a comparable T cell that does not comprise the modification(s) or wild-type T cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings are for illustration purposes only, not for limitation.

[0022] FIG. 1A, FIG. IB, FIG. 1C, and FIG. ID are each a series of graphs showing phenotypes of exemplary engineered human anti-CD19 CAR T cells, as assessed by flow cytometry for expression of each of HLA class I, HLA class II, CD47, CD 19 CAR, and FKBP-12 expression (shown in darker color) compared to respective isotype controls (shown in light gray color). FIG. 1A is a series of graphs showing phenotypes of exemplary wildtype (WT) CAR T cells. FIG. IB is a series of graphs showing phenotypes of exemplary hypoimmunogenic (HIP) CAR T cells. Fig. 1C is a series of graphs showing phenotypes of exemplary WT-FKBP knock-out (FKBPKO) CAR T cells. Fig. ID is a series of graphs showing phenotypes of exemplary HIP -FKBPKO CAR T cells.

[0023] FIG. 2A is a schematic providing an overview of an experimental protocol. Humanized mice were intravenously injected with 350,000 WT CAR T cells, WT-FKBPKO CAR T cells, HIP CAR T cells, or HIP -FKBPKO CAR T cells. After 6 days, spleens and sera were recovered.

[0024] FIG. 2B is a graph providing IFN-y spot frequency of exemplary WT or HIP CAR T cells with or without FKBPKO, as assessed by Elispot assays as described herein, (dashed line: background; bars: mean ± SD, 3 mice per group).

[0025] FIG. 2C is a graph showing levels of IgM donor-specific antibodies (DSAs) bound to exemplary CAR T cells in de-complemented mouse sera, as assessed by flow cytometry and expressed as mean fluorescence intensity (MFI), as described herein (dashed line: background; bars: mean ± SD, 3 mice per group).

[0026] FIG. 2D is a series of graphs showing normalized cell index of exemplary target T cells over time after introduction of allogeneic natural killer (NK) effector cells at an effectortarget (E:T) ratio of 1 : 1, as assessed by cytotoxicity assay as described herein. Results from exemplary target T cell types are shown in separate graphs and included untransduced (“Mock”) T cells, HLA class I and Il-depleted T cells (“DKO”), and WT and HIP CAR T cells with or without FKBP12 knockout (FKBP-KO) (mean ± SD, three replicates per group and time point).

[0027] FIG. 2E is a series of graphs showing normalized cell index of exemplary target T cells after introduction of allogeneic macrophage effector cells at an E:T ratio of 1 : 1, as assessed by cytotoxicity assay as described herein (mean ± SD, three replicates per group and time point). Conventions and conditions as in FIG. 2D.

[0028] FIG. 3A is a graph showing normalized cell index of target Nalm6 cancer cells over time in the absence of effector cells, as assessed by cytotoxicity assay.

[0029] FIG. 3B is a pair of graphs showing normalized cell index of target Nalm6 cancer cells over time after introduction of WT CAR T effector cells in 1 : 1 (top) and 7: 1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0030] FIG. 3C is a pair of graphs showing normalized cell index of target Nalm6 cancer cells over time after introduction of HIP CAR T effector cells in 1 : 1 (top) and 7: 1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0031] FIG. 3D and FIG. 3E are each a series of line graphs showing normalized cell index of target Nalm6 cancer cells over time after introduction of WT CAR T effector cells for exemplary immunosuppression conditions, as assessed by cytotoxicity assay. Exemplary immunosuppression conditions included a clinically relevant dose of 12.5 ng / mL tacrolimus (“Tac clin”), a high dose of 125 ng / mL tacrolimus (“Tac high”), a clinically relevant dose of lOnM (9.1 ng / mL) rapamycin (“Rapa clin”), a high dose of lOOnM (91.4 ng / mL) rapamycin (“Rapa high), clinical doses of both tacrolimus and rapamycin (“Tac+Rapa clin”), and high doses of both tacrolimus and rapamycin (“Tac+Rapa high”). Graphs show mean ± SD with three replicates per group and time point. FIG. 3D shows results for experiments with an E:T ratio of 1 : 1. FIG. 3E shows results for experiments using an E:T ratio of 7: 1.

[0032] FIG. 3F and FIG. 3G are each a series of graphs showing normalized cell index of target Nalm6 cancer cells over time after introduction of HIP CAR T effector cells for exemplary immunosuppression conditions, as assessed by cytotoxicity assay.Conventions and exemplary immunosuppression conditions as in FIG. 3D. FIG. 3F shows results for experiments with an E:T ratio of 1 : 1. FIG 3G shows results for experiments using an E:T ratio of 7:1.

[0033] FIG. 4A is a graph showing normalized cell index of target Nalm6 cancer cells over time in the absence of effector cells, as assessed by cytotoxicity assay.

[0034] FIG. 4B is a pair of graphs showing normalized cell index of target Nalm6 cancer cells over time after introduction of WT-FKBPKO CAR T effector cells in 1 : 1 (top) and 7:1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0035] FIG. 4C is a pair of graphs showing normalized cell index of target Nalm6 cancer cells over time after introduction of HIP-FKBPKO CAR T effector cells in 1 : 1 (top) and 7:1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0036] FIG. 4D and FIG. 4E are each a series of graphs showing normalized cell index of target Naim 6 tumor cells over time after introduction of WT-FKBPKO CAR T effector cells for exemplary immunosuppression conditions. Conventions and exemplary immunosuppression conditions as in FIG. 3D. FIG. 4D shows results for experiments with an E:T ratio of 1 : 1. FIG. 4E shows results for experiments using an E:T ratio of 7: 1.

[0037] FIG. 4F and FIG. 4G are each a series of graphs showing normalized cell index of target Nalm6 tumor cells over time after introduction of HIP-FKBPKO CAR T effector cells for exemplary immunosuppression conditions. Conventions and exemplary immunosuppression conditions as in FIG. 3D. FIG. 4F shows results for experiments with an E:T ratio of 1 : 1. FIG. 4G shows results for experiments using an E:T ratio of 7: 1.

[0038] FIG. 5A is a schematic providing an overview of an experimental protocol. Humanized mice were intravenously injected with 50,000 Luc+ Nalm6 tumor cells on day 0. Some animal groups then received daily intraperitoneal injections of Tac and / or Rapa and continued throughout the 25-day study. Some groups received intravenous injections of 350,000 WT, WT-FKBPKO, HIP, or HIP-FKBPKO CAR T cells on day 3.

[0039] FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G each provide a series of images of tumor growth over time and a graph quantifying tumor growth in photons per second, as assessed by bioluminescence imaging as described herein. FIG. 5B provides data for immunocompetent humanized mice treated with Naim 6 cells only (5 mice). FIG. 5C provides data for humanized mice treated with immunosuppressive agents Tac and Rapa and with Nalm6 cells (4 mice). FIG. 5D provides data for humanized mice treated with WT CAR T cells, immunosuppressive agents Tac and Rapa, and Nalm6 cells (5 mice). FIG. 5E provides data for humanized mice treated with HIP CAR T cells, immunosuppressive agents Tac and Rapa, and Nalm6 cells (5 mice). FIG. 5F provides data for humanized mice treatedwith WT-FKBPKO CAR T cells, immunosuppressive agents Tac and Rapa, and Nalm6 cells (5 mice). FIG. 5G provides data for humanized mice treated with HIP-FKBPKO CAR T cells, immunosuppressive agents Tac and Rapa, and Nalm6 cells (5 mice).

[0040] FIG. 6A is a schematic showing an experimental protocol. Spleen and bone marrow was recovered from mice of the in vivo study on day 25 or the day they dropped out of the study.

[0041] FIG. 6B is a graph showing fractions of CD 19+ cells in bone marrow for mice treated with Nalm6 cells only (“Nalm6 only”) compared to mice treated with Nalm6 cells and with both tacrolimus and rapamycin (“IS”), as assessed by flow cytometry (5 mice in Nalm6 only group; 4 mice in IS group; graphs show mean ± SD; groups were compared using the Mann-Whitney test).

[0042] FIG. 6C and FIG. 6D are graphs showing fractions of CAR+ cells in mice treated with immunosuppressive agents tacrolimus and rapamycin and with either WT- FKBPKO CAR T cells or HIP-FKBPKO CAR T cells, as assessed with flow cytometry (5 mice per group; graphs show mean ± SD; groups were compared using the Mann-Whitney test). FIG. 6C shows data from bone marrow cells. FIG. 6D shows data from spleen cells.

[0043] FIG. 6E is a graph showing fractions of CD 19+ cells in bone marrow for mice treated with immunosuppressive agents tacrolimus and rapamycin and with either WT- FKBPKO CAR T cells or HIP-FKBPKO CAR T cells, as assessed by flow cytometry (5 mice per group; graphs show mean ± SD; differences were compared using the Mann-Whitney test).

[0044] FIG. 6F and FIG. 6G are graphs showing fractions of CAR+ cells in mice treated with immunosuppressive agents tacrolimus and rapamycin and with either WT CAR T cells or HIP CAR T cell, as assessed by flow cytometry (5 mice per group; graphs show mean ± SD; groups were compared using the Mann-Whitney test). FIG. 6F shows data from bone marrow cells. FIG. 6G shows data from spleen cells.

[0045] FIG. 6H is a graph showing fractions of CD 19+ cells in bone marrow for mice treated with immunosuppressive agents tacrolimus and rapamycin and with either WT CAR T cells or HIP CAR T cells (5 mice per group graphs show mean ± SD; groups were compared using the Mann -Whitney test).

[0046] FIG. 7A is a graph showing normalized cell index of target human B cells over time in the absence of effector cells, as assessed by cytotoxicity assay.

[0047] FIG. 7B is a pair of graphs showing normalized cell index of target human B cells over time after introduction of WT CAR T effector cells in 1 : 1 (top) and 7: 1 (bottom) E:T ratios in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0048] FIG. 7C is a pair of graphs showing normalized cell index of target human B cells over time after introduction of HIP CAR T effector cells in 1 : 1 (top) and 7: 1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0049] FIG. 7D and FIG. 7E are each a series of graphs showing normalized cell index of target human B cells over time after introduction of WT CAR T effector cells for exemplary immunosuppression conditions, as assessed by cytotoxicity assay. Exemplary immunosuppression conditions included a clinically relevant dose of 12.5 ng / mL tacrolimus (“Tac clin”), a high dose of 125 ng / mL tacrolimus (“Tac high”), a clinically relevant dose of lOnM (9.1 ng / mL) rapamycin (“Rapa clin”), a high dose of lOOnM (91.4 ng / mL) rapamycin (“Rapa high), clinical doses of both tacrolimus and rapamycin (“Tac+Rapa clin”), and high doses of both tacrolimus and rapamycin (“Tac+Rapa high”). Graphs show mean ± SD with three replicates per group and time point. FIG. 7D shows results for experiments with an E:T ratio of 1 : 1. FIG 7E shows results for experiments using an E:T ratio of 7: 1.

[0050] FIG. 7F and FIG. 7G are each a series of graphs showing normalized cell index of target human B cells over time after introduction of HIP CAR T effector cells for exemplary immunosuppression conditions, as assessed by cytotoxicity assay. Conventions and exemplary immunosuppression conditions as in FIG. 7D. FIG. 7F shows results for experiments with an E:T ratio of 1 : 1. FIG. 7F shows results for experiments using an E:T ratio of 7: 1.

[0051] FIG. 8A is a graph showing normalized cell index of target human B cells over time in the absence of effector cells, as assessed by cytotoxicity assay.

[0052] FIG. 8B is a pair of graphs showing normalized cell index of target human B cells over time after introduction of WT-FKBPKO CAR T effector cells in 1 : 1 (top) and 7: 1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0053] FIG. 8C is a pair of graphs showing normalized cell index of target human B cells over time after introduction of HIP-FKBPKO CAR T effector cells in 1 : 1 (top) and 7: 1 (bottom) E:T ratios and in the absence of immunosuppressive agents, as assessed by cytotoxicity assay.

[0054] FIG. 8D and FIG. 8E are each a series of graphs showing normalized cell index of target human B cells over time after introduction of WT-FKBPKO CAR T effector cells for exemplary immunosuppression conditions, as assessed by cytotoxicity assay. Conventions and exemplary immunosuppression conditions as in FIG. 7D. FIG. 8D shows results for experiments with an E:T ratio of 1 : 1. FIG. 8E shows results for experiments using an E:T ratio of 7: 1.

[0055] FIG. 8F and FIG. 8G are each a series of graphs showing normalized cell index of target human B cells over time after introduction of HIP-FKBPKO CAR T effector cells for exemplary immunosuppression conditions, as assessed by cytotoxicity assay. Conventions and exemplary immunosuppression conditions as in FIG. 7D. FIG. 8F shows results for experiments with an E:T ratio of 1 : 1. FIG. 8G shows results for experiments using an E:T ratio of 7: 1.

[0056] FIG. 9A is a schematic showing an experimental protocol. Blood was drawn from all mice of the in vivo study that were alive on day 25.

[0057] FIG. 9B and FIG. 9C are graphs showing trough levels of exemplary immunosuppressive agents in serum or whole blood for exemplary mouse treatment groups, as assessed by ELISA. Graphs show mean ± SD (5 mice per group). FIG. 9B shows trough levels of tacrolimus. FIG. 9C shows trough levels of rapamycin.DETAILED DESCRIPTIONI. INTRODUCTION

[0058] Described herein are engineered or modified immunosuppression-resistant cells. In some embodiments, immunosuppression-resistant cells are engineered T cells. In some embodiments, an engineered T cell comprises one or more modifications that reduce expression of FKBP12 relative to a comparable T-cell which does not comprises the modification(s) or relative to a wild-type T cell. In some embodiments, an engineered T cell comprises one or more modifications that reduce expression of cyclophilin A relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell. Advantageously, such cells can be resistant to immunosuppressive agents such as tacrolimus and / or rapamycin. Accordingly, in some embodiments, immunosuppression-resistant T cells may be used in the treatment of a subject receiving immunosuppressive therapy, e.g., a patient with a post-transplant or autoimmune disorder.

[0059] In some embodiments, immunosuppression-resistant cells described herein are further engineered or modified to express a CAR.

[0060] In some embodiments, immunosuppression-resistant cells described herein are further engineered or modified to evade the immune system. Accordingly, described herein are engineered or modified immune evasive cells, including, but not limited to, human immune evasive cells. To overcome the problem of a subject's immune rejection of these primary and / or stem cell-derived transplants, the inventors have developed and describe herein hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent cells, differentiated cells derived from such, and primary cells) that represent a viable source for any transplantable cell type. Such cells are protected from adaptive and / or innate immune rejection upon administration to a recipient subject. Advantageously, the hypoimmunogenic cells disclosed herein are not rejected by the recipient subject's immune system, regardless of the subject's genetic make-up, as they are protected from adaptive and innate immune rejection upon administration to a recipient subject. In some embodiments, the engineered and / or hypoimmunogenic cells do not express major histocompatibility complex (MHC) class I and class II antigens and / or T-cell receptors. In some embodiments, the engineered and / or hypoimmunogenic cells do not express MHC I and II antigens and / or T-cell receptors and overexpress CD47 proteins. In some embodiments, the engineered and / or hypoimmunogenic cells such as engineered and / or hypoimmunogenic T cells do not express MHC I and II antigens and / or T-cell receptors, overexpress CD47 proteins and express exogenous CARs.

[0061] In some embodiments, hypoimmunogenic cells described herein are not subject to an innate immune cell rejection. In some embodiments, hypoimmunogenic cells are not susceptible to NK cell-mediated lysis. In some embodiments, hypoimmunogenic cells are not susceptible to macrophage engulfment. In some embodiments, hypoimmunogenic cells are useful as a source of universally compatible cells.

[0062] In some embodiments, the technology disclosed herein utilizes expression of tolerogenic factors and modulation (e.g., reduction or elimination) of FKBP12, cyclophilin A, MHC I, MHC II, and / or TCR expression in human cells. In some embodiments, genome editing technologies utilizing rare-cutting endonucleases (e.g., CRISPR / Cas) are also used to reduce or eliminate expression of genes involved in an immune response (e.g., by deleting genomic DNA of genes involved in an immune response or by insertions of genomic DNA into such genes, such that gene expression is impacted) in the cells. In some embodiments, genome editing technologies or other gene modulation technologies are used to inserttolerance-inducing (tolerogenic) factors in human cells, rendering the cells and their progeny (include any differentiated cells prepared therefrom) able to evade immune recognition upon engrafting into a recipient subject. As such, the cells described herein exhibit modulated expression of one or more genes and factors that affect MHC I, MHC II, and / or TCR expression and evade the recipient subject’s immune system.

[0063] The genome editing techniques enable double-strand DNA breaks at desired locus sites. These controlled double-strand breaks promote homologous recombination at the specific locus sites. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequences and induce a double-stranded break in the nucleic acid molecule. The doublestrand break is repaired either by an error-prone non-homologous end-joining (NHEJ) or by homologous recombination (HR).

[0064] Accordingly, in some embodiments, an engineered T cell comprises modifications that (a) reduce expression of FKBP12 and / or cyclophilin A, (b) reduce expression of: (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules, (c) increase expression of a tolerogenic factor, and (d) express a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modification(s) or relative to a wild-type T cell.

[0065] Described herein are methods of treating or preventing a disorder comprising administering to a subject a composition comprising immunosuppression resistant cells. In some embodiments, a subject has, is diagnosed as having, or is suspected of having a posttransplant disorder as described herein. In some embodiments, a subject has, is diagnosed as having, or is suspected of having an autoimmune disorder as described herein. In some embodiments, a subject is receiving an immunosuppressive therapy.

[0066] The present disclosure also describes pharmaceutical compositions comprising a population of immunosuppression-resistant cells as described herein, as well as methods of making a population of immunosuppression-resistant cells as described herein. Also described herein are methods comprising administering to a subject a composition comprising a population of immunosuppression-resistant cells.

[0067] The practice of the numerous embodiments will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley -Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perb al, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in Immunology.II. DEFINITIONS

[0068] As described in the present disclosure, the following terms will be employed, and are defined as indicated below.

[0069] The term "autoimmune disease" or “autoimmune disorder” or “inflammatory disease” or “inflammatory disorder” refers to any disease or disorder in which the subject mounts an immune response against its own tissues and / or cells. Autoimmune disorders can affect almost any organ system in the subject (e.g., human), including, but not limited to, diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to Hashimoto's thyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis, Myasthenia gravis and Diabetes.

[0070] The term "cancer" as used herein is defined as a hyperproliferation of cells whose unique trait (e.g., loss of normal controls) results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. With respect to the inventive methods, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and / or urinary bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissues of the malignant type, unless otherwise specifically indicated and does not include a benign type tissue.

[0071] As used herein, “clinically effective amount” refers to an amount sufficient to provide a clinical benefit in the treatment and / or management of a disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount that has been shown to produce at least one improved clinical endpoint to the standard of care for the disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount that has been demonstrated, for example in a clinical trial, to be sufficient to provide statistically significant and meaningful effectiveness for treating the disease, disorder, or condition. In some embodiments, the clinically effective amount is also a therapeutically effective amount. In other embodiments, the clinically effective amount is not a therapeutically effective amount.

[0072] In some embodiments, an alteration or modification (including, for example, genetic alterations or modifications) described herein results in reduced expression of a target or selected polynucleotide sequence. In some embodiments, an alteration or modification described herein results in reduced expression of a target or selected polypeptide sequence. In some embodiments, an alteration or modification described herein results in increasedexpression of a target or selected polynucleotide sequence. In some embodiments, an alteration or modification described herein results in increased expression of a target or selected polypeptide sequence.

[0073] The terms "decrease," "reduced," "reduction," and "decrease" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, decrease," "reduced," "reduction," "decrease" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10- 100% as compared to a reference level. In some embodiments, the cells are engineered to have reduced expression of one or more targets relative to an unaltered or unmodified wildtype cell.

[0074] The term “donor subject” refers to an animal, for example, a human from whom cells can be obtained. The term “donor subject” also encompasses animals, for example, a human, from whom an organ is obtained for transplant in a recipient subject. The “non-human animals” and “non -human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “donor subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the donor subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like. A “donor subject” can also refer to more than one donor, for example one or more humans or non- human animals or non-human mammals.

[0075] As used herein, the team “encode” or “encoding” refers to a first molecule that is produced by a second molecule, wherein the sequence information of the second molecule determines the sequence of the first molecule. For example, a first molecule can have a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids which are determined by the sequence of the second molecule (e.g., a polynucleotide). For example, a DNA molecule (e.g., a second molecule) can encode an RNA molecule (e.g., a second molecule, for example by a transcription process that includes a DNA-dependent RNA polymerase enzyme) or a polypeptide (e.g., a first molecule for example by a transcription and a translation process). An RNA molecule (e.g., a second molecule) can encode a polypeptide (e.g., a first molecule forexample by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system.

[0076] The term "endogenous" refers to a referenced molecule or polypeptide that is naturally present in the cell. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid naturally contained within the cell and not exogenously introduced. Similarly, the term when used in reference to a promoter sequence refers to a promoter sequence naturally contained within the cell and not exogenously introduced.

[0077] The term "engineered cell" as used herein refers to a cell that has been altered in at least some way by human intervention, including, for example, by genetic alterations or modifications such that the engineered cell differs from a wild-type cell.

[0078] As used herein, the term "exogenous" in the context of a polynucleotide or polypeptide being expressed is intended to mean that the referenced molecule or the referenced polypeptide is introduced into the cell of interest. The polypeptide can be introduced, for example, by introduction of an encoding nucleic acid into the genetic material of the cells such as by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. An exogenous polynucleotide can be inserted into at least one allele of the cell using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus based viral vector. In some embodiments, the exogenous polynucleotide is inserted into a safe harbor or target locus of at least one allele of the cell.

[0079] An "exogenous" molecule is a molecule, construct, factor and the like that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods. "Normal presence in the cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, amolecule that is present only during embryonic development of neurons is an exogenous molecule with respect to an adult neuron cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.

[0080] An exogenous molecule or factor can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or doublestranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA- binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.

[0081] An exogenous molecule or construct can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. In such instances, the exogenous molecule is introduced into the cell at greater concentrations than that of the endogenous molecule in the cell. In some instances, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE- dextran-mediated transfer and viral vector-mediated transfer.

[0082] A "gene," for the purposes of the present disclosure, includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and / or locus control regions.

[0083] Gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and / or glycosylation.

[0084] The term “genetic modification” and its grammatical equivalents as used herein can refer to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome. For example, genetic modification can refer to alterations, additions, and / or deletion of genes or portions of genes or other nucleic acid sequences. A genetically modified cell can also refer to a cell with an added, deleted and / or altered gene or portion of a gene. A genetically modified cell can also refer to a cell with an added nucleic acid sequence that is not a gene or gene portion. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms, and mechanisms that result in permanent knock-in, knock-down, or knock-out of target genes or portions of genes or nucleic acid sequences Genetic modifications include, for example, both transient knock-in and mechanisms that result in permanent knock-in of nucleic acids sequences Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

[0085] As used herein, the terms "grafting", "administering," "introducing", "implanting" and "transplanting" as well as grammatical variations thereof are used interchangeably in the context of the placement of cells (e.g., cells described herein) into a subject, by a method or route which results in localization or at least partial localization of the introduced cells at a desired site or systemic introduction (e.g., into circulation). The cells can be implanted directly to the desired site, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e. g. twenty -four hours, to a few days, to as long as several years. In some embodiments, the cells can also be administered (e.g., injected) a location other than the desired site, such as in the brain or subcutaneously, for example, in a capsule to maintain the implanted cells at the implant location and avoid migration of the implanted cells.

[0086] By "HLA" or "human leukocyte antigen" complex is a gene complex encoding the Major Histocompatibility Complex (MHC) proteins in humans. These cell-surface proteins that make up the HLA complex are responsible for the regulation of the immune response to antigens. In humans, there are two MHCs, class I and class II, "HLA-I" and "HLA-II". HLA-I includes three proteins, HLA- A, HLA-B and HLA-C, which present peptides from the inside of the cell, and antigens presented by the HLA-I complex attract killer T-cells (also known as CD8+ T-cells or cytotoxic T cells). The HLA-I proteins are associated with P-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA- DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates CD4+ cells (also known as T-helper cells). It should be understood that the use of either "MHC" or "HLA" is not meant to be limiting, as it depends on whether the genes are from humans (HLA). Thus, as it relates to mammalian cells, these terms may be used interchangeably herein.

[0087] As used herein to characterize a cell, the term "hypoimmunogenic" generally means that such cell is less prone to innate or adaptive immune rejection by a subject into which such cells are transplanted, e.g., the cell is less prone to allorejection by a subject into which such cells are transplanted. For example, relative to a cell of the same cell type that does not comprise the modifications, such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to innate or adaptive immune rejection by a subject into which such cells are transplanted. In some embodiments, genome editing technologies are used to modulate the expression of MHC I and MHC II genes, and thus, contribute to generation of a hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell evades immune rejection in an MHC- mismatched allogeneic recipient. In some embodiments, a hypoimmunogenic cell is protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection. Detailed descriptions of hypoimmunogenic cells, methods of producing thereof, and methods of using thereof are found in W02016183041 filed May 9, 2015;WO2018132783 filed January 14, 2018; WO2018175390 filed March 20, 2018; W02020018615 filed July 17, 2019; W02020018620 filed July 17, 2019; PCT / US2020 / 44635 filed July 31, 2020; WO2021022223 filed July 31, 2020; W02021041316 filed August 24, 2020; WO2021222285 filed April 27, 2021, 2020; and WO2021222285 filed April 27, 2021, the disclosures including the examples, sequence listings and figures are incorporated herein by reference in their entirety.

[0088] Hypoimmunogenicity of a cell can be determined by evaluating the immunogenicity of the cell such as the cell’s ability to elicit adaptive and innate immune responses or to avoid eliciting such adaptive and innate immune responses. Such immune response can be measured using assays recognized by those skilled in the art. In some embodiments, an immune response assay measures the effect of a hypoimmunogenic cell on T cell proliferation, T cell activation, T cell killing, donor specific antibody generation, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, hypoimmunogenic cells and derivatives thereof undergo decreased killing by T cells and / or NK cells upon administration to a subject. In some instances, the cells and derivatives thereof show decreased macrophage engulfment compared to an unmodified or wild-type cell. In some embodiments, a hypoimmunogenic cell elicits a reduced or diminished immune response in a recipient subject compared to a corresponding unmodified wild-type cell. In some embodiments, a hypoimmunogenic cell is nonimmunogenic or fails to elicit an immune response in a recipient subject.

[0089] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0090] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0091] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0092] Immune signaling factor" as used herein refers to, in some cases, a molecule, protein, peptide and the like that activates immune signaling pathways.

[0093] "Immunosuppressive factor" or "immune regulatory factor" or "tolerogenic factor" as used herein include hypoimmunity factors, complement inhibitors, and other factors that modulate or affect the ability of a cell to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment. These may be in combination with additional genetic modifications.

[0094] The terms "increased", "increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In some embodiments, the reference level, also referred to as the basal level, is 0.

[0095] In some embodiments, the alteration is an indel. As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. A gene editing (e.g., CRISPR / Cas) system of the present disclosure can be used to induce an indel of any length or a point mutation in a target polynucleotide sequence.

[0096] As used herein, the term “inhibitor” refers to an agent whose presence or level correlates with decreased level or activity of a target to be modulated. In some embodiments, an inhibitor may act directly (in which case it exerts its influence directly upon its target, for example by binding to the target); in some embodiments, an inhibitor may act indirectly (in which case it exerts its influence by interacting with and / or otherwise altering a regulator of a target, so that level and / or activity of the target is reduced). In some embodiments, an inhibitor is one whose presence or level correlates with a target level or activity that is reduced relative to a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known inhibitor, or absence of the inhibitor as disclosed herein, etc.). For example, in some embodiments, an inhibitor is an FKBP inhibitor, whose presence is correlated with a decreased level or activity of a FK506- binding protein, e.g. a decreased level of FKBP12. In some embodiments, an inhibitor is a cyclophilin inhibitor whose presence is correlated with a decreased level or activity of a cyclophilin, e.g. a decreased level of cyclophilin A.

[0097] As used herein, “knock down” refers to a reduction in expression of the target mRNA or the corresponding target protein. Knock down is commonly reported relative to levels present following administration or expression of a noncontrol molecule that does not mediate reduction in expression levels of RNA (e.g., a non-targeting control shRNA, siRNA, or miRNA). In some embodiments, knock down of a target gene is achieved by genetic modification, including use of gene editing systems (e.g., CRISPR / Cas).

[0098] Knock down is commonly assessed by measuring the mRNA levels using quantitative polymerase chain reaction (qPCR) amplification or by measuring protein levels by western blot or enzyme-linked immunosorbent assay (ELISA). Analyzing the protein level provides an assessment of both mRNA cleavage as well as translation inhibition. Further techniques for measuring knock down include RNA solution hybridization, nuclease protection, northern hybridization, gene expression monitoring with a microarray, antibody binding, radioimmunoassay, and fluorescence activated cell analysis. Those skilled in the art will readily appreciate how to use the gene editing systems (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

[0099] By "knock in" or “knock-in” herein is meant a genetic modification resulting from the insertion of a DNA sequence into a chromosomal locus in a host cell. This causes initiation of or increased levels of expression of the knocked in gene, portion of gene, ornucleic acid sequence inserted product, e.g., an increase in RNA transcript levels and / or encoded protein levels. As will be appreciated by those in the art, this can be accomplished in several ways, including inserting or adding one or more additional copies of the gene or portion thereof to the host cell or altering a regulatory component of the endogenous gene increasing expression of the protein is made or inserting a specific nucleic acid sequence whose expression is desired. This may be accomplished by modifying a promoter, adding a different promoter, adding an enhancer, adding other regulatory elements, or modifying other gene expression sequences.

[0100] As used herein, "knock out" or “knock-out” includes deleting all or a portion of a target polynucleotide sequence in a way that interferes with the translation or function of the target polynucleotide sequence. For example, a knock out can be achieved by altering a target polynucleotide sequence by inducing an insertion or a deletion (“indel”) in the target polynucleotide sequence, including in a functional domain of the target polynucleotide sequence (e.g., a DNA binding domain). Those skilled in the art will readily appreciate how to use the gene editing systems (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

[0101] In some embodiments, a genetic modification or alteration results in a knock out or knock down of the target polynucleotide sequence or a portion thereof. Knocking out a target polynucleotide sequence or a portion thereof using a gene editing system (e.g., CRISPR / Cas) of the present disclosure can be useful for a variety of applications. For example, knocking out a target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in a cell ex vivo and introducing those cells comprising the knocked out mutant allele into a subject) or for changing the genotype or phenotype of a cell.

[0102] "Modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation may also be complete, i.e., wherein gene expression is totally inactivated or is activated to wild-type levels or beyond; or it may be partial, wherein gene expression is partially reduced, or partially activated to some fraction of wild-type levels.

[0103] The methods provided herein can be used to alter a target polynucleotide sequence in a cell. The present disclosure contemplates altering target polynucleotide sequences in a cell for any purpose. In some embodiments, the target polynucleotide sequence in a cell is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell with a resulting genotype that differs from its original genotype. In some instances, a "mutant cell" exhibits a mutant phenotype, for example when a normally functioning gene is altered using the gene editing systems (e.g., CRISPR / Cas) systems of the present disclosure. In other instances, a "mutant cell" exhibits a wild-type phenotype, for example when a gene editing system (e.g., CRISPR / Cas) system of the present disclosure is used to correct a mutant genotype. In some embodiments, the target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, the target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0104] The term “native cell” as used herein refers to a cell that is not otherwise modified (e.g., engineered). In some embodiments, a native cell is a naturally occurring wildtype or a control cell.

[0105] The term “objective response rate” or “ORR” is defined as a proportion of subjects with a best overall response of complete response or partial response as determined by an investigator and / or central assessment. Subjects who do not meet the criteria for an objective response by an analysis cutoff date will be considered non-responders. In some embodiments, disease assessments obtained after infusion and up through an observation of progression or start of new anti -cancer therapy will be used. In some embodiments, response assessment in non-Hodgkin lymphoma subjects will be based on the Lugano classification criteria and in chronic lymphocytic leukemia subjects based on the International Workshop on Chronic Lymphocytic Leukemia criteria.

[0106] The term "operatively linked" or "operably linked" are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptionalregulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.

[0107] As used herein, "promoter," "promoter sequence," or "promoter region" refers to a DNA regulatory region / sequence capable of binding RNA polymerase and involved in initiating transcription of a downstream coding or non-coding sequence. In some examples, the promoter sequence includes the transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. In some embodiments, the promoter sequence includes a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes.

[0108] In some embodiments, the engineered and hypoimmunogenic cells described are propagated from a primary T cell or a progeny thereof. As used herein, the term “propagated from a primary T cell or a progeny thereof’ encompasses the initial primary T cell that is isolated from the donor subject and any subsequent progeny thereof. As used herein, the term “progeny” encompasses, e.g., a first-generation progeny, i.e., the progeny is directly derived from, obtained from, obtainable from or derivable from the initial primary T cell by, e.g., traditional propagation methods. The term “progeny” also encompasses further generations such as second, third, fourth, fifth, sixth, seventh, or more generations, i.e., generations of cells which are derived from, obtained from, obtainable from or derivable from the former generation by, e.g., traditional propagation methods. The term “progeny” also encompasses modified cells that result from the modification or alteration of the initial primary T cell or a progeny thereof.

[0109] The term “recipient patient” refers to an animal, for example, a human to whom treatment, including prophylactic treatment, with the cells as described herein, is provided. The term “recipient patient” also encompasses animals, for example, humans who have received or will receive an organ transplant or allograft. For treatment of those infections, conditions or disease states, which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. The term “recipient patient” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the recipient patient is a mammal such as a human, orother mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like.

[0110] As used herein, the terms "regulatory sequences," "regulatory elements," and "control elements" are interchangeable and refer to polynucleotide sequences that are upstream (5' non-coding sequences), within, or downstream (3' non-translated sequences) of a polynucleotide target to be expressed. Regulatory sequences influence, for example but are not limited to, the timing of transcription, amount or level of transcription, RNA processing or stability, and / or translation of the related structural nucleotide sequence. Regulatory sequences may include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translational initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like. It is recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleotide sequences of different lengths may have identical regulatory or promoter activity.[OHl] As used herein, a “target” can refer to a gene, a portion of a gene, a portion of the genome, or a protein that is subject to regulatable reduced expression by the methods described herein.

[0112] As used herein, “therapeutically effective amount” refers to an amount sufficient to provide a therapeutic benefit in the treatment and / or management of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount sufficient to ameliorate, palliate, stabilize, reverse, slow, attenuate or delay the progression of a disease, disorder, or condition, or of a symptom or side effect of the disease, disorder, or condition. In some embodiments, the therapeutically effective amount is also a clinically effective amount. In other embodiments, the therapeutically effective amount is not a clinically effective amount.

[0113] As used herein, the term "treating" and "treatment" includes administering to a subject a therapeutically or clinically effective amount of cells described herein so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired therapeutic or clinical results. For purposes of this technology, beneficial or desired therapeutic or clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a condition, disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the condition, disease or disorder.

[0114] For purposes of this technology, beneficial or desired therapeutic or clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0115] A "vector" or "construct" is capable of transferring gene sequences to target cells. Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors. Methods for the introduction of vectors or constructs into cells are known to those of skill in the art and include, but are not limited to, lipid- mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and / or viral vector-mediated transfer.

[0116] In some embodiments, cells of the present disclosure are engineered to have reduced or increased expression of one or more targets relative to an unaltered or unmodified wild-type cell. In some embodiments, cells of the present disclosure are engineered to have constitutive reduced or increased expression of one or more targets relative to an unaltered or unmodified wild-type cell. In some embodiments, cells of the present disclosure are engineered to have regulatable reduced or increased expression of one or more targets relative to an unaltered or unmodified wild-type cell. In some embodiments, cells of the present disclosure comprise decreased expression of FKBP12 relative to a wild-type cell or a comparable cell of the same type. In some embodiments, the cells comprise increased expression of CD47 relative to a wild-type cell or a comparable cell of the same cell type. By “wild-type” or “wt” or “control” in the context of a cell means any cell found in nature.Examples of wild type or control cells include primary cells and T cells found in nature. However, by way of example, in the context of an engineered cell, as used herein, “wildtype” or “control” or “comparable” can mean an engineered cell that may contain nucleic acid changes resulting in reduced expression of MHC I and / or II and / or T-cell receptors, but did not undergo the gene editing procedures to result in for example, overexpression of CD47 proteins, expression of a CAR, reduction in FKBP12 expression, and / or reduction in cyclophilin A expression. For example, as used herein, “wild-type” or “control” or “comparable” means an engineered cell that comprises reduced or knocked out expression of B2M, CIITA, and / or TRAC. Also as used herein, “wild-type” or “control” or “comparable” means an engineered cell that comprises reduced or knocked out expression of B2M, CIITA, TRAC, and / or TRBC. As used herein, “wild-type” or “control” or “comparable” also means an engineered cell that may contain nucleic acid changes resulting in overexpression of CD47 proteins, but did not undergo the gene editing procedures to result in reduced expression of MHC I and / or II and / or T-cell receptors. As used herein, “wild-type” or “control” or “comparable” also means an engineered cell that may contain nucleic acid changes resulting in overexpression of CD47 proteins, expression of a CAR, reduction in FKBP12 expression, and / or reduction in cyclophilin A expression, but did not undergo the gene editing procedures to result in reduced expression of MHC I and / or II and / or T-cell receptors. In some embodiments, the cells are engineered to have regulatable reduced or increased expression of one or more targets relative to a cell of the same cell type that does not comprise the modifications. In some embodiments, the wild-type cell or the control cell is a starting material. In some embodiments, the starting material is a primary cell collected from a donor. In some embodiments, the starting material is a primary blood cell collected from a donor, e.g., via a leukopak. For example, unmodified T cells obtained from a donor is a starting material that are considered wild-type or control cells as contemplated herein. In another example, an iPSC cell line starting material is a starting material that is considered a wildtype or control cell as contemplated herein. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cell.

[0117] It is noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure, representative illustrative methods and materials are now described.

[0118] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number, which, in the context presented, provides the substantial equivalent of the specifically recited number. The term about is used herein to mean plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110.

[0119] All publications, patents, and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated herein by reference to disclose and describe the subject matter in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the technology described herein is not entitled to antedate such publication by virtue of prior technology. Further, the dates ofpublication provided might be different from the actual publication dates, which may need to be independently confirmed.

[0120] Before the technology is further described, it is to be understood that this technology is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. It should also be understood that the headers used herein are not limiting and are merely intended to orient the reader, but the subject matter generally applies to the technology disclosed herein.III. DETAILED DESCRIPTION OF THE EMBODIMENTSPost-Transplant Disorders

[0121] Solid organ transplantation is a life-saving treatment for patients with terminal disease or with end-organ dysfunction which significantly impairs quality of life. Solid organ transplantation is possible in large part due to the advent of immunosuppressive agents such as cyclosporine. Autologous CAR T cell therapy in solid organ transplant recipients on lifelong systemic immunosuppression has so far been disappointing. The mTOR inhibitors tacrolimus (Tac) and rapamycin (Rapa) are among the most common immunosuppressive drugs and while they prevent organ allograft rejection, they also suppress the efficacy and persistence of a CAR T product.Antibody-Mediated Rejection (AMR)

[0122] The emergence of graft-directed antibodies often precedes subsequent graft rejection and graft failure. Consequently, patients who develop graft-directed antibodies have a high probability of graft failure. In turn, graft failure can have serious implications for patient survival.

[0123] Current treatment strategies for AMR include preventing B cell activation / differentiation, enhancing the formation of regulatory T cells, and depleting alloantibody-producing plasma cells. Specific treatments for AMR include co-stimulation blockers such as Belatacept. There is no approved effective treatment for treating antibody- mediated rejection. The difficulty of reducing or preventing graft-directed antibodies in antibody-mediated rejection is further complicated by treatments required to prevent othertypes of allograft rejection, such as immunosuppressive regimens comprising one or more immunosuppressive agents.

[0124] B cell-targeting CAR T cells might help with controlling AMR. In this scenario, reduction of immunosuppression during ongoing AMR may be particularly risky. Therefore, CAR T cells resistant to immunosuppression might offer advantages for patients that depend on immunosuppression to protect their transplanted organ. In addition, an allogeneic cell product would further allow the use of healthy donor T cells not taken from an immunosuppressed cancer patient.

[0125] In some embodiments, the present disclosure is directed to a novel approach for treating antibody -mediated rejection wherein the production of graft-directed antibodies is reduced by directly targeting B cells and / or plasma cells that release such antibodies. Accordingly, disclosed herein is a method of treating antibody -mediated rejection comprising administering to a subject a composition comprising immunosuppression-resistant T cells. In some embodiments, CAR T cells deplete B cells in a subject. In some embodiments, administration of CAR T cells reduces a level of graft-directed antibodies in a subject.Post-Transplant Lymphoproliferative Disorder (PTLD)

[0126] The risk for developing lymphoproliferative disease is increased in immunocompromized patients and those that are on long-term immunosuppression. The latter include solid organ transplant recipients and their risk for post-transplant lymphoproliferative disorder (PTLD) has been shown to correlate with the level of immunosuppression. Heart or lung transplant recipients remain on relatively high doses of immunosuppressants even years after the transplantation and up to 20% of long-term surviving patients will be diagnosed with PTLD. Patients after kidney and liver transplantation require less immunosuppression and only 1-5% will acquire PTLD. Most PTLD cases are of B cell origin (> 85-90%) and two thirds are associated with Epstein Barr Virus (EBV). The standard of care for transplant patients with PTLD has traditionally involved reduction of immunosuppression and rituximab-based chemotherapy, but more recently, small case series on CAR T cell therapy have been reported. Although CAR T cell therapy has shown remarkable efficacy in non-transplant patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL), its use in transplant patients is jeopardized by immunosuppression. Besides the desired effect to suppress host T cells to prevent rejection, CAR T cell function is also inhibited and their anti-tumor activity impaired. Therefore,immunosuppression is usually reduced before leukapheresis to allow the extraction of functionally recovered autologous T cells for the manufacturing of the CAR T cell product. After the CAR T cell infusion, a balance act is necessary to keep the level of immunosuppression as low as possible to enable CAR T cell killing of the tumor, while still protecting the allograft from rejection. Since long-term persistence of functionally active CAR T cells has been associated with durable remission in leukemia, the management of immunosuppression will remain a life-long challenge in these patients.

[0127] In some embodiments, the present disclosure is directed to a novel approach for treating PTLD, wherein cancerous B cells are reduced by directly targeting B cells and / or plasma cells. Accordingly, disclosed herein is a method of treating PTLD comprising administering to a subject a composition comprising immunosuppression-resistant cells. In some embodiments, CAR T cells deplete B cells in a subject.A. Genetically Modified Cells i. Immunosuppression-Resistant Cells

[0128] Immunophilins are a highly conserved superfamily of peptidyl-prolyl isomerases (PPIs) believed to chaperone protein folding. The two major families of immunophilins are cyclophilins (Cyps) and FK506-binding proteins (FKBPs). Functionally, these two families of immunophilins are defined by their binding to specific immunosuppressant molecules, with Cyps binding to cyclosporin A, and FKBPs binding to FK506 (tacrolimus) and / or rapamycin. The immunosuppressant molecules to which immunophilins characteristically bind are of fungal origin. The binding of these molecules to immunophilins is associated with inhibition of PPIs which in turn suppresses immune activity.

[0129] Multiple input signal in T cells converge at mTOR and make it an important integrator controlling different T cell functions. TCR engagement rapidly activates mTOR and the magnitude of activation correlates with the duration of the T cell-dentritic cell interaction and the dose of the cognate antigen. CD28- and OX40-co-stimulation, often used in CAR constructs, further enhance the mTOR signaling via PI3K-AKT activation. The inhibition of mTOR inhibits T cell proliferation and promotes the induction of anergy. Tacrolimus and rapamycin inhibit T cell activation and proliferation and can preventengraftment and persistence of wild-type and hypoimmunogenic CAR T-cells in immunosuppressed subjects.

[0130] In some embodiments, the present disclosure is directed to primary cells (such as, but not limited to, primary T cells). In some embodiments, primary cells such as primary T cells are engineered for reduced expression or lack of expression of immunophilins. In some embodiments, primary cells such as primary T cells include a genomic modification of the FKBP1A gene. In some embodiments, primary cells such as primary T-cells are engineered for reduced expression or lack of expression of FKBP12 (FKBP1 A). In some embodiments, primary cells such as primary T cells include a genomic modification of the Peptidylprolyl Isomerase A (PPIA) gene. In some embodiments, primary cells such as primary T-cells are engineered for reduced expression or lack of expression of cyclophilin A.

[0131] In some embodiments, such immunosuppression-resistant T cells and primary T cells express a chimeric antigen receptor (CAR). In some embodiments, a CAR comprises an antigen binding domain that binds to a ligand expressed on B cells, plasma cells and / or plasmablasts. In some embodiments, a CAR is or comprises a CDlO-specific CAR, a CD 19- specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD24-specific CAR, a CD27- specific CAR, a CD38-specific CAR, a CD45R-specific CAR, a CD138-specific CAR, a CD319-specific CAR, or a BCMA-specific CAR. In some embodiments, a CAR is or comprises a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a BCMA- specific CAR, a GPRC5D-specific CAR, a CD38-specific CAR, a CD70-specific CAR, a CD79b-specific CAR, or an EBV antigen-specific CAR. In some embodiments, a CAR comprises an antigen binding domain that binds to CD 19, CD20, CD22, BCMA, GPRC5D, CD27, CD30, EBNA1, LMP1, LMP2, LMP2A, EBNA3A, EBNA3C, BZLF1, BMLF1, gp350, or gH / gL. In some embodiments, a CAR is a CD19-specific CAR.

[0132] In some embodiments, cells are modified or engineered relative to a comparable cell or a wild-type cell, including an unaltered or unmodified wild-type cell or control cell. In some embodiments, a comparable or wild-type cell is a starting material. In some embodiments, a starting material is a primary cell collected from a donor. In some embodiments, a starting material is a primary blood cell collected from a donor, e.g., via a leukopak. In some embodiments, a starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cell.

[0133] In some embodiments, immunosuppression-resistant cells of the present disclosure are resistant against calcineurin inhibitors and mTOR inhibitors as both types of inhibitors depend on FKBP12 signaling. In some embodiments, immunosuppressionresistant cells of the present disclosure maintain their efficacy even in a subject treated with high levels of immunosuppressive therapy.

[0134] In some embodiments, immunosuppression-resistant cells as disclosed herein can be used in a method for treating or preventing a post-transplant disorder. In some embodiments, a post-transplant disorder comprises antibody -mediated rejection (AMR). In some embodiments, a post-transplant disorder comprises a post-transplant lymphoproliferative disorder (PTLD). In some embodiments, immunosuppression-resistant cells as disclosed herein can be used in a method for treating or preventing an autoimmune disorder. ii. Hypoimmunogenic Cells

[0135] Provided herein are immunosuppressant resistant cells which are further engineered for reduced expression or lack of expression of MHC class I and / or MHC class II human leukocyte antigens, and in some instances, for reduced expression or lack of expression of a T-cell receptor (TCR) complex. In some embodiments, such hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR) in addition to reduced expression or lack of expression of MHC class I and / or MHC class II human leukocyte antigens, and have reduced expression or lack expression of a T-cell receptor (TCR) complex. In some embodiments, a CAR targets a B cell antigen. In some embodiments, a CAR is a CD19-specific CAR. In some embodiments, cells are modified or engineered as compared to a wild-type or control cell, including an unaltered or unmodified wild-type cell or control cell. In some embodiments, a wild-type cell or the control cell is a starting material. In some embodiments, a starting material is a primary cell collected from a donor. In some embodiments, a starting material is a primary blood cell collected from a donor, e.g., via a leukopak. In some embodiments, a starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cell. In some embodiments, hypoimmunogenic, immunosuppression-resistant engineered cells of the present disclosure are more efficacious in subjects with suppressed immune systems relative to comparable cells. In some embodiments, hypoimmunogenic, immunosuppression-resistant engineered cells of the present disclosure persist in subjects with suppressed immune systems longer than comparable cells. In some embodiments, hypoimmunogenic, immunosuppression-resistant engineered cells of the present disclosure enhance depletion of bone marrow CD 19 cells in subjects with suppressed immune systems relative to comparable cells. In some embodiments, hypoimmunogenic, immunosuppression-resistant engineered cells of the present disclosure completely deplete bone marrow CD 19 cells (e.g., levels of bone marrow CD 19 cells are undetectable) in subjects with suppressed immune systems. In some embodiments, the comparable cells are HIP CAR T cells that are not immunosuppression-resistant.

[0136] In some embodiments, immunosuppressant resistant engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR) and include a genomic modification of the B2M gene. In some embodiments, immunosuppressant resistant cells engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and include a genomic modification of the CIITA gene. In some embodiments, immunosuppressant resistant cells engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR, and include a genomic modification of the TRAC gene. In some embodiments, immunosuppressant resistant cells engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR, and include one or more genomic modifications selected from the group consisting of the B2M, CIITA, and TRAC genes. In some embodiments, immunosuppressant resistant cells engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR, and include genomic modifications of the B2M, CIITA, and TRAC genes. In some embodiments, the cells are B2M- / -, CIITA- / -, TRAC- / - cells that express a CD47 transgene (CD47tg) and a CAR. In some embodiments, immunosuppressant resistant cells engineered and / or hypoimmune (HIP) T cells are produced by differentiating induced pluripotent stem cells such as engineered and / or hypoimmunogenic induced pluripotent stem cells.

[0137] In certain embodiments, the cells are B2Mindel / indel, CIITA"ldcl"ldcl, TRACindel / indel, CD47tg cells that also express CARs. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Thl cells, Th2 cells, Th9 cells, Thl7 cells, T-follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tern) cells, effector memory T cells express CD45RA (TEMRAcells), tissue-resident memory (Trm) cells, virtual memory T cells, innate memory T cells, memory stem cell (Tsc), y5 T cells, and any other subtype of T cells. In some embodiments, primary T cells are selected from a group that includes cytotoxic T-cells, helper T-cells, memory T-cells, regulatory T-cells, tumor infiltrating lymphocytes, and combinations thereof. In some embodiments, cells are modified or engineered as compared to a wild-type or control cell, including an unaltered or unmodified wild-type cell or control cell. In some embodiments, a wild-type cell or a control cell is a starting material. In some embodiments, starting material is a primary cell collected from a donor. In some embodiments, starting material is a primary blood cell collected from a donor, e.g., via a leukopak. In some embodiments, starting material is otherwise modified or engineered to have altered expression of one or more genes to generate an engineered cell.

[0138] In some embodiments, primary T cells are from a pool of primary T cells from one or more donor subjects that are different than the recipient subject (e.g., the patient administered the cells). Primary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. Primary T cells can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10, or more 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, primary T cells are harvested from one or a plurality of individuals, and in some instances, primary T cells or a pool of primary T cells are cultured in vitro. In some embodiments, primary T cells or a pool of primary T cells are engineered to exogenously express CD47 and cultured in vitro.

[0139] In certain embodiments, primary T cells or a pool of primary T cells are engineered to express a chimeric antigen receptor (CAR). Useful CARs include those that bind a B cell antigen, e.g. a CD 19 antigen. In some cases, a CAR is the same or equivalent to those used in FDA-approved CAR-T cell therapies such as, but not limited to, those used in tisagenlecleucel and axicabtagene ciloleucel, or others under investigation in clinical trials.

[0140] In some embodiments, the primary T cells or the pool of primary T cells are engineered to exhibit reduced expression of an endogenous T cell receptor compared to unmodified primary T cells. Methods of genetically modifying a cell including a T cell are described in detail, for example, in US2023 / 0272429, US2022 / 0331358, US2024 / 0252642, WO2023 / 287827, W02020 / 018620 and W02016 / 183041, the disclosures of which are herein incorporated by reference in their entireties, including the tables, appendices, sequence listing and figures.

[0141] In some embodiments, CAR-T cells comprise a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains.

[0142] In some embodiments, CAR-T cells comprise a CAR comprising an antigen binding domain, a transmembrane, and one or more signaling domains. In some embodiments, the CAR also comprises a linker. In some embodiments, the CAR comprises a CD 19 antigen binding domain. In some embodiments, the CAR comprises a CD8a transmembrane domain. In some embodiments, the CAR comprises a CD8a signal peptide. In some embodiments, the CAR comprises a Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 24). In some embodiments, the antigen binding domain of the CAR is selected from a group including, but not limited to, (a) an antigen binding domain targets an antigen characteristic of a neoplastic cell or an antigen binding domain targets an antigen characteristic of an autoimmune disorder.

[0143] In some embodiments, the CAR further comprises one or more linkers. The format of an scFv is generally two variable domains linked by a flexible peptide sequence, or a “linker,” either in the orientation VH-linker-VL or VL-linker-VH. Any suitable linker known to those in the art in view of the specification can be used in the CARs. Examples of suitable linkers include, but are not limited to, a GS based linker sequence, and a Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 24).

[0144] In some embodiments, the antigen binding domain is selected from a group that includes an antibody, an antigen-binding portion or fragment thereof, an scFv, and a Fab. In some embodiments, the antigen binding domain binds to CD 19. In some embodiments, the antigen binding domain is an anti -CD 19 scFv such as but not limited to FMC63.

[0145] In some embodiments, the transmembrane domain comprises one selected from a group that includes a transmembrane region of TCRa, TCRP, TCR^, CD3s, CD3y, CD35, CD3< CD4, CD5, CD8a, CD8P, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcsRIy, VEGFR2, FAS, FGFR2B, and functional variant thereof. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain.

[0146] In some embodiments, the signaling domain(s) of the CAR comprises a costimulatory domain(s). For instance, a signaling domain can contain a costimulatory domain. Or, a signaling domain can contain one or more costimulatory domains. In certainembodiments, the signaling domain comprises a costimulatory domain. In other embodiments, the signaling domains comprise costimulatory domains. In some cases, when the CAR comprises two or more costimulatory domains, two costimulatory domains are not the same. In some embodiments, the costimulatory domains comprise two costimulatory domains that are not the same. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the costimulatory domains enhance cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation.

[0147] In some embodiments, a CAR comprises a CD3 zeta (CD3Q intracellular domain or functional variant thereof. In some embodiments, a CAR comprises a 4-1BB costimulatory intracellular domain or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3(^ domain or functional variant thereof; and (ii) a 4- IBB domain or functional variant thereof.

[0148] Methods for introducing a CAR construct or producing CAR-T cells are well known to those skilled in the art. Detailed descriptions are found, for example, in Vormittag et al., Curr Opin Biotechnol, 2018, 53, 162-181; and Eyquem et al., Nature, 2017, 543, 113- 117.

[0149] In some embodiments, the cells derived from primary T cells comprise reduced expression of an endogenous T cell receptor, for example by disruption of an endogenous T cell receptor gene (e.g., T cell receptor alpha constant region (TRAC)).

[0150] In some embodiments, a CD47 transgene is inserted into a random locus of a cell. In some embodiments, a transgene encoding a CAR is inserted into a random locus of a cell. In some embodiments, a transgene encoding a CAR is inserted into a random locus of a cell via viral vector transduction / integration. In some embodiments, a CD47 transgene and a transgene encoding a CAR are inserted into a random locus of a cell via viral vector transduction / integration. In some embodiments, a vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope. In some embodiments, a transgene encoding a CAR is inserted into at least one allele of a cell using viral transduction. In some embodiments, an exogenous polynucleotide is inserted into at least one allele of a cell using a lentivirus based viral vector.

[0151] In some embodiments, a CD47 transgene and a transgene encoding a CAR are inserted into the same locus.

[0152] In certain embodiments, a CD47 and a CAR are controlled by a single promoter and are encoded by a single transgene. In some instances, the promoter controlling expression of any transgene described is a constitutive promoter. In other instances, the promoter for any transgene described is an inducible promoter. In some embodiments, the promoter is an EFla promoter. In some embodiments, the promoter is CAG promoter. In some embodiments, a CD47 transgene and a transgene encoding a CAR are both controlled by a constitutive promoter. In some embodiments, a CD47 transgene and a transgene encoding a CAR are both controlled by an inducible promoter.

[0153] Methods provided are useful for inactivation or ablation of MHC class I expression and / or MHC class II expression in cells such as primary T cells. In some embodiments, genome editing technologies utilizing rare-cutting endonucleases (e.g., CRISPR / Cas) are also used to reduce or eliminate expression of genes involved in an immune response (e.g., by deleting genomic DNA of genes involved in an immune response or by insertions of genomic DNA into such genes, such that gene expression is impacted) in cells. In certain embodiments, genome editing technologies or other gene modulation technologies are used to insert tolerance-inducing factors in human cells, rendering them and the differentiated cells prepared therefrom hypoimmunogenic cells. As such, the hypoimmunogenic cells have reduced or eliminated expression of MHC I and MHC II expression. In some embodiments, the cells are nonimmunogenic (e.g., do not induce an innate and / or an adaptive immune response) in a recipient subject.

[0154] In some embodiments, a cell includes a modification to increase expression of CD47.

[0155] In some embodiments, a cell comprises a genomic modification of one or more target polynucleotide sequences that regulate the expression of either MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules. In some embodiments, a genetic editing system is used to modify one or more target polynucleotide sequences. In some embodiments, the targeted polynucleotide sequence is one or more selected from the group including B2M and CIITA. In some embodiments, the cell comprises a genetic editing modification to the B2M gene. In some embodiments, the cell comprises a genetic editing modification to the CIITA gene. In some embodiments, the cell comprises genetic editing modifications to the B2M and CIITA genes. In certain embodiments, the genome of the cell has been altered to reduce or delete critical components of HLA expression. In some embodiments, the cells are modified or engineered as comparedto a wild-type or control cell, including an unaltered or unmodified wild-type cell or control cell. In some embodiments, the wild-type cell or the control cell is a starting material. In some embodiments, the starting material is a primary cell collected from a donor. In some embodiments, the starting material is a primary blood cell collected from a donor, e.g., via a leukopak. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cell.

[0156] In some embodiments, the present disclosure provides a cell (e.g., primary T cell or CAR-T cell) or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell (e.g., primary T cell or CAR-T cell) or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In numerous embodiments, the present disclosure provides a cell (e.g., primary T cell or CAR-T cell) or population thereof comprising a genome in which one or more genes has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cell or population thereof.

[0157] In certain embodiments, the expression of MHC I molecules and / or MHC II molecules is modulated by targeting and deleting a contiguous stretch of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M and CIITA. In some embodiments, described herein are genetically edited cells (e.g., modified human cells) comprising exogenous CD47 proteins and inactivated or modified CIITA gene sequences, and in some instances, additional gene modifications that inactivate or modify B2M gene sequences.

[0158] Provided herein are cells exhibiting a modification of one or more targeted polynucleotide sequences that regulates the expression of any one of the following: (a) MHC I antigens, (b) MHC II antigens, (c) TCR complexes, (d) both MHC I and II antigens, and (e) MHC I and II antigens and TCR complexes. In certain embodiments, the modification includes increasing expression of CD47. In some embodiments, the cells include an exogenous or recombinant CD47 polypeptide. In certain embodiments, the modification includes expression of a chimeric antigen receptor. In some embodiments, the cells comprise an exogenous or recombinant chimeric antigen receptor polypeptide.

[0159] In some embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of TCR molecules in the cell or population thereof. In numerous embodiments, the present disclosure provides a cell or population thereof comprising a genome in which one or more genes has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules and TCR complex molecules in the cell or population thereof.

[0160] In some embodiments, the cells and methods described herein include genomically editing human cells to cleave CIITA gene sequences as well as editing the genome of such cells to alter one or more additional target polynucleotide sequences such as, but not limited to, B2M and TRAC. In some embodiments, the cells and methods described herein include genomically editing human cells to cleave B2M gene sequences as well as editing the genome of such cells to alter one or more additional target polynucleotide sequences such as, but not limited to, CIITA and TRAC. In some embodiments, the cells and methods described herein include genomically editing human cells to cleave TRAC gene sequences as well as editing the genome of such cells to alter one or more additional target polynucleotide sequences such as, but not limited to, B2M and CIITA.

[0161] In some embodiments, a population of engineered cells described evades NK cell mediated cytotoxicity upon administration to a recipient patient. In some embodiments, a population of engineered cells evades NK cell mediated cytotoxicity by one or more subpopulations of NK cells. In some embodiments, a population of engineered is protected from cell lysis by NK cells, including immature and / or mature NK cells upon administration to a recipient patient. In some embodiments, a population of engineered cells evades macrophage engulfment upon administration to a recipient patient. In some embodiments, apopulation of engineered cells does not induce an innate and / or an adaptive immune response to the cell upon administration to a recipient patient.

[0162] In some embodiments, cells described herein are controlled by a safety switch. The term “safety switch” used herein refers to a system for controlling the expression of a gene or protein of interest that, when downregulated or upregulated, leads to clearance or death of the cell, e.g., through recognition by the host’s immune system. A safety switch can be designed to be triggered by an exogenous molecule in case of an adverse clinical event. A safety switch can be engineered by regulating the expression on the DNA, RNA and protein levels. A safety switch includes a protein or molecule that allows for the control of cellular activity in response to an adverse event. In some embodiments, a safety switch comprises a therapeutic agent that inhibits or blocks the interaction of CD47 and SIRPa. In some aspects, the CD47-SIRPa blockade agent is an agent that neutralizes, blocks, antagonizes, or interferes with the cell surface expression of CD47, SIRPa, or both. In some embodiments, the CD47-SIRPa blockade agent inhibits or blocks the interaction of CD47, SIRPa or both. In some embodiments, a CD47-SIRPa blockade agent (e.g., a CD47-SIRPa blocking, inhibiting, reducing, antagonizing, neutralizing, or interfering agent) comprises an agent selected from a group that includes an antibody or fragment thereof that binds CD47, a bispecific antibody that binds CD47, an immunocytokine fusion protein that bind CD47, a CD47 containing fusion protein, an antibody or fragment thereof that binds SIRPa, a bispecific antibody that binds SIRPa, an immunocytokine fusion protein that bind SIRPa, an SIRPa containing fusion protein, and a combination thereof.

[0163] In some embodiments, the population of engineered cells described elicits a reduced level of immune activation or no immune activation upon administration to a recipient subject. In some embodiments, the cells elicit a reduced level of systemic TH1 activation or no systemic TH1 activation in a recipient subject. In some embodiments, the cells elicit a reduced level of immune activation of peripheral blood mononuclear cells (PBMCs) or no immune activation of PBMCs in a recipient subject. In some embodiments, the cells elicit a reduced level of donor-specific IgG antibodies or no donor specific IgG antibodies against the cells upon administration to a recipient subject. In some embodiments, the cells elicit a reduced level of IgM and IgG antibody production or no IgM and IgG antibody production against the cells in a recipient subject. In some embodiments, the cells elicit a reduced level of cytotoxic T cell killing of the cells upon administration to a recipient subject.1. CIITA

[0164] In some embodiments, the technologies disclosed herein modulate (e.g., reduces or eliminates) the expression of MHC II genes by targeting and modulating (e.g., reducing or eliminating) Class II transactivator (CIITA) expression. In some embodiments, the modulation occurs using a CRISPR / Cas system. CIITA is a member of the LR or nucleotide binding domain (NBD) leucine-rich repeat (LRR) family of proteins and regulates the transcription of MHC II by associating with the MHC enhanceosome.

[0165] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of CIITA. In some embodiments, the target polynucleotide sequence is a homolog of CIITA. In some embodiments, the target polynucleotide sequence is an ortholog of CIITA.

[0166] In some embodiments, reduced or eliminated expression of CIITA reduces or eliminates expression of one or more of the following MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0167] In some embodiments, the cells described herein comprise gene modifications at the gene locus encoding the CIITA protein. In other words, the cells comprise a genetic modification at the CIITA locus. In some instances, the nucleotide sequence encoding the CIITA protein is set forth in RefSeq. No. NM_000246.4 and NCBI Genbank No. U18259. In some instances, the CIITA gene locus is described in NCBI Gene ID No. 4261. In certain cases, the amino acid sequence of CIITA is depicted as NCBI GenBank No. AAA88861.1. Additional descriptions of the CIITA protein and gene locus can be found in Uniprot No. P33076, HGNC Ref. No. 7067, and OMIM Ref. No. 600005.

[0168] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the CIITA gene. In some embodiments, the genetic modification targeting the CIITA gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOS:5184-36352 of Table 12 of W02016183041, which is herein incorporated by reference. In some embodiments, the cell has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject. In someembodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CIITA gene.

[0169] Assays to test whether the CIITA gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CIITA gene by PCR and the reduction of HLA-II expression can be assays by FACS analysis. In another embodiment, CIITA protein expression is detected using a Western blot of cells lysates probed with antibodies to the CIITA protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus based viral vector.2. B2M

[0170] In some embodiments, the technologies disclosed herein modulate (e.g., reduce or eliminate) the expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of the accessory chain B2M. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) expression of B2M, surface trafficking of MHC-I molecules is blocked and the cell rendered hypoimmunogenic. In some embodiments, the cell has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject.

[0171] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

[0172] In some embodiments, decreased or eliminated expression of B2M reduces or eliminates expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0173] In some embodiments, the cells described herein comprise gene modifications at the gene locus encoding the B2M protein. In other words, the cells comprise a genetic modification at the B2M locus. In some instances, the nucleotide sequence encoding the B2M protein is set forth in RefSeq. No. NM_004048.4 and Genbank No. AB021288.1. In some instances, the B2M gene locus is described in NCBI Gene ID No. 567. In certain cases, the amino acid sequence of B2M is depicted as NCBI GenBank No. BAA35182.1. Additional descriptions of the B2M protein and gene locus can be found in Uniprot No. P61769, HGNC Ref. No. 914, and OMIM Ref. No. 109700.

[0174] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the B2M gene. In some embodiments, the genetic modification targeting the B2M gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOS:81240-85644 of Table 15 of W02016183041, which is herein incorporated by reference. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the B2M gene. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus based viral vector.

[0175] Assays to test whether the B2M gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the B2M gene by PCR and the reduction of HLA-I expression can be assays by FACS analysis. In another embodiment, B2M protein expression is detected using a Western blot of cells lysates probedwith antibodies to the B2M protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification.3. TRAC

[0176] In many embodiments, the technologies disclosed herein modulate (e.g., reduce or eliminate) the expression of TCR genes including the TRAC gene by targeting and modulating (e.g., reducing or eliminating) expression of the constant region of the T cell receptor alpha chain. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) expression of TRAC, surface trafficking of TCR molecules is blocked. In some embodiments, the cell also has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject.

[0177] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRAC. In some embodiments, the target polynucleotide sequence is a homolog of TRAC. In some embodiments, the target polynucleotide sequence is an ortholog of TRAC.

[0178] In some embodiments, decreased or eliminated expression of TRAC reduces or eliminates TCR surface expression.

[0179] In some embodiments, the cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells comprise gene modifications at the gene locus encoding the TRAC protein. In other words, the cells comprise a genetic modification at the TRAC locus. In some instances, the nucleotide sequence encoding the TRAC protein is set forth in Genbank No. X02592.1. In some instances, the TRAC gene locus is described in RefSeq. No. NG_001332.3 and NCBI Gene ID No. 28755. In certain cases, the amino acid sequence of TRAC is depicted as Uniprot No. P01848. Additional descriptions of the TRAC protein and gene locus can be found in Uniprot No. P01848, HGNC Ref. No. 12029, and OMIM Ref. No. 186880.

[0180] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the TRAC gene. In some embodiments, the genetic modification targeting the TRAC gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acidsequence for specifically targeting the TRAC gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene is selected from the group consisting of SEQ ID NOS: 532-609 and 9102-9797 of US20160348073, which is herein incorporated by reference.

[0181] Assays to test whether the TRAC gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the TRAC gene by PCR and the reduction of TCR expression can be assays by FACS analysis. In another embodiment, TRAC protein expression is detected using a Western blot of cells lysates probed with antibodies to the TRAC protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification.4. TRB

[0182] In many embodiments, the technologies disclosed herein modulate (e.g., reduce or eliminate) the expression of TCR genes including the gene encoding T cell antigen receptor, beta chain (e.g., the TRB, TRBC, or TCRB gene) by targeting and modulating (e.g., reducing or eliminating) expression of the constant region of the T cell receptor beta chain. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) expression of TRB, surface trafficking of TCR molecules is blocked. In some embodiments, the cell also has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject.

[0183] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRB. In some embodiments, the target polynucleotide sequence is a homolog of TRB. In some embodiments, the target polynucleotide sequence is an ortholog of TRB.

[0184] In some embodiments, decreased or eliminated expression of TRB reduces or eliminates TCR surface expression.

[0185] In some embodiments, the cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells comprise gene modifications at the gene locus encoding the TRB protein. In other words, the cells comprise a genetic modification at the TRB gene locus. In some instances, the nucleotide sequence encoding theTRB protein is set forth in UniProt No. P0DSE2. In some instances, the TRB gene locus is described in RefSeq. No. NG_001333.2 and NCBI Gene ID No. 6957. In certain cases, the amino acid sequence of TRB is depicted as Uniprot No. P01848. Additional descriptions of the TRB protein and gene locus can be found in GenBank No. L36092.2, Uniprot No. P0DSE2, and HGNC Ref. No. 12155.

[0186] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the TRB gene. In some embodiments, the genetic modification targeting the TRB gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRB gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRB gene is selected from the group consisting of SEQ ID NOS:610-765 and 9798-10532 of US20160348073, which is herein incorporated by reference.

[0187] Assays to test whether the TRB gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the TRB gene by PCR and the reduction of TCR expression can be assays by FACS analysis. In another embodiment, TRB protein expression is detected using a Western blot of cells lysates probed with antibodies to the TRB protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification.

[0001] In many embodiments, the technologies disclosed herein modulate (e.g., reduce or eliminate) the expression of TCR genes including the gene encoding T cell antigen receptor, beta chain (e.g., the TRB, TRBC, or TCRB gene) by targeting and modulating (e.g., reducing or eliminating) expression of the constant region of the T cell receptor beta chain. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) expression of TRB, surface trafficking of TCR molecules is blocked. In some embodiments, the cell also has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject.

[0188] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRB. In some embodiments, the target polynucleotide sequence is a homolog of TRB. In some embodiments, the target polynucleotide sequence is an ortholog of TRB.

[0189] In some embodiments, decreased or eliminated expression of TRB reduces or eliminates TCR surface expression.

[0190] In some embodiments, the cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells comprise gene modifications at the gene locus encoding the TRB protein. In other words, the cells comprise a genetic modification at the TRB gene locus. In some instances, the nucleotide sequence encoding the TRB protein is set forth in UniProt No. P0DSE2. In some instances, the TRB gene locus is described in RefSeq. No. NG_001333.2 and NCBI Gene ID No. 6957. In certain cases, the amino acid sequence of TRB is depicted as Uniprot No. P01848. Additional descriptions of the TRB protein and gene locus can be found in GenBank No. L36092.2, Uniprot No.P0DSE2, and HGNC Ref. No. 12155.

[0191] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the TRB gene. In some embodiments, the genetic modification targeting the TRB gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRB gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRB gene is selected from the group consisting of SEQ ID NOS:610-765 and 9798-10532 of US20160348073, which is herein incorporated by reference.

[0192] Assays to test whether the TRB gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the TRB gene by PCR and the reduction of TCR expression can be assays by FACS analysis. In another embodiment, TRB protein expression is detected using a Western blot of cells lysates probed with antibodies to the TRB protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification.5. Tolerogenic Factors

[0193] In many embodiments, one or more tolerogenic factors can be inserted or reinserted into genome-edited cells to create immune-privileged universal donor cells, such as universal donor stem cells, universal donor T cells, or universal donor cells. In certainembodiments, the hypoimmunogenic cells disclosed herein have been further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, without limitation, one or more of CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl -Inhibitor, IL- 10, IL-35, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and Serpinb9. In some embodiments, the tolerogenic factors are selected from the group consisting of CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4- Ig, IL-10, IL-35, FasL, Serpinb9, CCL21, CCL22, and Mfge8. In some embodiments, the tolerogenic factors are selected from the group consisting of DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, Cl -inhibitor, and IL-35. In some embodiments, the tolerogenic factors are selected from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, Cl -inhibitor, and IL-35. In some embodiments, the tolerogenic factors are selected from a group including CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and Serpinb9.

[0001] In some embodiments, the polynucleotide encoding the one or more tolerogenic factors is inserted into at least one allele of the T cell using viral transduction. In some embodiments, the polynucleotide encoding the one or more tolerogenic factors is inserted into at least one allele of the T cell using a lentivirus based viral vector. In some embodiments, the lentivirus based viral vector is a pseudotyped, self-inactivating lentiviral vector that carries the polynucleotide encoding the one or more tolerogenic factors. In some embodiments, the lentivirus based viral vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the polynucleotide encoding the one or more tolerogenic factors.CD47

[0194] In some embodiments, the present disclosure provides a cell or population thereof that has been modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide) CD47. In some embodiments, the present disclosure provides a method for altering a cell genome to express CD47. In some embodiments, the cell expresses exogenous CD47. In some instances, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some embodiments, the cell is genetically modified to comprise an integrated exogenous polynucleotide encoding CD47 usinghomology-directed repair. In some instances, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus. In some instances, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide wherein the nucleotide sequence is inserted into at least one allele of an AAVS1 locus. In some instances, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide wherein the nucleotide sequence is inserted into at least one allele of an CCR5 locus. In some instances, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide wherein the nucleotide sequence is inserted into at least one allele of a safe harbor or target gene locus, such as, but not limited to, a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBL gene locus, a Rosa gene locus, an F3 (CD 142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1 (CD91) gene locus, a HMGB1 gene locus, an ABO gene locus, an RHD gene locus, a FUT1 locus, and a KDM5D gene locus. In some instances, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide wherein the nucleotide sequence is inserted into at least one allele of a TRAC locus.

[0195] CD47 is a leukocyte surface antigen and has a role in cell adhesion and modulation of integrins. It is expressed on the surface of a cell and signals to circulating macrophages not to eat the cell.

[0196] In some embodiments, the cell outlined herein comprises a nucleotide sequence encoding a CD47 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cell outlined herein comprises a nucleotide sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cell comprises a nucleotide sequence for CD47 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM 198793.2. In some embodiments, the cell comprises a nucleotide sequence for CD47 as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2. In some embodiments, the nucleotide sequence encoding a CD47 polynucleotide is a codon optimized sequence. In some embodiments, the nucleotide sequence encoding a CD47 polynucleotide is a human codon optimized sequence.

[0197] In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cell outlined herein comprises a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1.

[0198] Exemplary amino acid sequences of human CD47 with a signal sequence and without a signal sequence are provided in Table 2.Table 2. Amino acid sequences of human CD47

[0199] In some embodiments, the cell comprises a CD47 polypeptide having at least95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cell comprises a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the cell comprises a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 86.

[0200] In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%,or more) to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 86. In some embodiments, the nucleotide sequence is codon optimized for expression in a particular cell.

[0201] In some embodiments, the CD47 is an engineered CD47. In some embodiments, the cell comprises a nucleotide sequence encoding an engineered CD47. In some embodiments, the cell expresses an engineered CD47 polypeptide on the surface of the cell. Examples of engineered CD47 variants can be found in WO2023158836 and PCT / US2024 / 043615, herein incorporated by reference.

[0202] In some embodiments, a suitable gene editing system (e.g., CRISPR / Cas system or any of the gene editing systems described herein) is used to facilitate the insertion of a polynucleotide encoding CD47, into a genomic locus of the hypoimmunogenic cell. In some cases, the polynucleotide encoding CD47 is inserted into a safe harbor or target locus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D gene locus. In some embodiments, the polynucleotide encoding CD47 is inserted into a B2M gene locus, a CIITA gene locus, a TRAC gene locus, or a TRB gene locus. In some embodiments, the polynucleotide encoding CD47 is inserted into any one of the gene loci provided herein. In certain embodiments, the polynucleotide encoding CD47 is operably linked to a promoter.

[0203] In some embodiments, the polynucleotide encoding CD47 is inserted into at least one allele of the T cell using viral transduction. In some embodiments, the polynucleotide encoding CD47 is inserted into at least one allele of the T cell using a lentivirus based viral vector. In some embodiments, the lentivirus based viral vector is a pseudotyped, self-inactivating lentiviral vector that carries the polynucleotide encoding CD47. In some embodiments, the lentivirus based viral vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the polynucleotide encoding CD47.

[0204] In another embodiment, CD47 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CD47 protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the exogenous CD47 mRNA.B. Chimeric Antigen Receptors

[0205] Provided herein are immunosuppression-resistant cells comprising a chimeric antigen receptor (CAR). CARs (also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give host cells (e.g., T cells) the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. In some embodiments, a CAR may comprise an extracellular binding domain (also referred to as a “binder”) that specifically binds a target antigen, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and / or one or more intracellular costimulatory domains.Domains may be directly adjacent to one another, or there may be one or more amino acids linking the domains.

[0206] A nucleotide sequence encoding a CAR may be derived from a mammalian sequence, for example, a mouse sequence, a primate sequence, a human sequence, or combinations thereof. In the cases where a nucleotide sequence encoding a CAR is nonhuman, the sequence of the CAR may be humanized. A nucleotide sequence encoding a CAR may also be codon-optimized for expression in a mammalian cell, for example, a human cell. In any of these embodiments, a nucleotide sequence encoding a CAR may be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein. Sequence variations may be due to codon-optimalization, humanization, restriction enzyme-based cloning scars, and / or additional amino acid residues linking the functional domains, etc.

[0207] In some embodiments, a CAR binds to a B cell antigen. In some embodiments, a CAR binds to CD19. In some embodiments, the CAR is a second generationCAR. In some embodiments, the CAR includes a single binding domain that binds to a single target antigen.

[0208] In some embodiments, a CD 19 specific CAR includes an anti-CD19 singlechain antibody fragment (scFv), a transmembrane domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3(^ signaling domain.

[0209] In some embodiments, a CAR comprises a commercial CAR construct carried by a T cell. Non-limiting examples of commercial CAR-T cell based therapies include brexucabtagene autoleucel (TEC ARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®), Descartes-08 and Descartes- 11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CYAD-211 from Clyad Oncology.

[0210] In some embodiments, a hypoimmunogenic cell described herein comprises a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, a hypoimmunogenic cell described herein comprises a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the polynucleotide is or comprises a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and at least one signaling domain (e.g., one, two or three signaling domains). In some embodiments, the CAR comprises a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains. In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, the antigen binding domain is or comprises an antibody, an antibody fragment, an scFv or a Fab.1. Antigen binding domain (ABD)

[0211] In some embodiments, an extracellular binding domain of a CAR may comprise one or more antibodies specific to one target antigen or multiple target antigens. An antibody may be an antibody fragment, for example, an scFv, or a single-domain antibody fragment, for example, a VHH. In some embodiments, an antigen binding domain comprises a light chain amino acid sequence and a heavy chain amino acid sequence. In some embodiments, a light chain amino acid sequence is not a full-length light chain sequence, but does comprise a light chain variable domain sequence. In some embodiments, a heavy chain amino acid sequence is not a full-length heavy chain sequence, but does comprise a light chain variable domain sequence. In certain embodiments, the scFv may comprise a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody connected by a linker. The VH and the VL may be connected in either order, i.e., VH-linker-VL or VL- linker-VH. Non-limiting examples of linkers include Whitlow linker, (G4S)n (n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.) linker, and variants thereof. In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain.

[0212] In some embodiments, the antigen binding domain (ABD) targets an antigen or ligand characteristic of a B cell. In some embodiments, the ABD targets an antigen or ligand characteristic of a healthy B cell. In some embodiments, the antigen binding domain targets an antigen characteristic of a neoplastic cell. In other words, in some embodiments, the antigen binding domain targets an antigen expressed by a neoplastic or cancer cell. In some embodiments, the ABD binds a tumor associated antigen. In some embodiments, the antigen characteristic of a neoplastic cell (e.g., antigen associated with a neoplastic or cancer cell) or a tumor associated antigen is CD 19 or an antigenic fragment or antigenic portion thereof.

[0213] In some embodiments, the antigen binding domain targets an antigen characteristic of a Bell cell mediated autoimmune disorder. In some embodiments, the ABD binds an antigen associated with a B cell mediated autoimmune disorder. In some instances, the antigen is expressed by a cell associated with a B cell mediated autoimmune disorder. In some embodiments, the a B cell mediated autoimmune disorder is selected from the group consisting of: systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, anti- neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, multiple sclerosis, pemphigus vulgaris, autoimmune blistering skin diseases, membranous nephropathy, anti -NMD A receptor neuropathy,neuromyelitis optica, idiopathic thrombocytopenic purpura, autoimmune hepatitis, type 1 diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy, polymyositis / dermatomyositis, stiff persons disease, anti- NMDA receptor encephalitis, anti-synthetase autoimmune syndromes, anti-phospholipid antibody syndrome, Sjogren’s syndrome, cryoglobulinemia, focal segmental glomerulosclerosis, rapidly progressive glomerulopathy, and autoimmune hemolytic anemia.

[0214] In some embodiments, an antigen binding domain of a CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, an antigen binding domain of a CAR binds to CD 10, CD 19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, GPRC5D, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2. See, e.g., US 2003 / 0077249; WO 2017 / 058753; WO 2017 / 058850, the contents of which are herein incorporated by reference.

[0215] In some embodiments, a CAR antigen binding domain is or comprises an antibody or an antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, a CAR antigen binding domain comprises an scFv or Fab fragment of a CD 19 antibody.2. Transmembrane domain

[0216] In some embodiments, a CAR of the present disclosure comprises a transmembrane domain comprising a CD8a transmembrane domain or functional variant thereof. Table 3 provides the amino acid sequences of exemplary transmembrane domains.Table 3. Exemplary sequences of transmembrane domains3. Intracellular signaling domain

[0217] In some embodiments, a CAR described herein comprises two intracellular signaling domains, wherein the two intracellular signaling domains are or comprise a CD3(^ signaling domain (or a functional fragment thereof) and 4- IBB co-stimulatory domain (or a functional fragment thereof). Table 4 provides amino acid sequences few exemplary intracellular costimulatory and / or signaling domains. In certain embodiments, as in the case of tisagenlecleucel as described below, the CD3(^ signaling domain of SEQ ID NO: 18 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14 (see SEQ ID NO:3).Table 4. Exemplary sequences of intracellular costimulatory and / or signaling domains4. Additional CAR elements

[0218] In some embodiments, a CAR comprises a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD8a signal peptide, IgK signal peptide, and granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GMCSFR-a, also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 5 below.Table 5. Exemplary sequences of signal peptides

[0219] In certain embodiments, a CAR may comprise a hinge domain, also referred to as a spacer. The terms “hinge” and “spacer” may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 6 below.Table 6. Exemplary sequences of hinge domains5. CD19 CARs

[0220] In some embodiments, a CAR is a CD 19 CAR (“CD 19-CAR”). In some embodiments, a CD 19 CAR comprises a signal peptide, an extracellular binding domain that specifically binds CD 19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain in tandem.

[0221] In some embodiments, a CD 19 CAR signal peptide comprises a CD8a signal peptide. In some embodiments, a CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:6. In some embodiments, a signal peptide comprises an IgK signal peptide. In some embodiments, an IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:7. In some embodiments, a signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, a GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:8.

[0222] In some embodiments, a CD 19 CAR extracellular binding domain is specific to CD 19, for example, human CD 19. A CD 19 CAR extracellular binding domain can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0223] In some embodiments, the extracellular binding domain of a CD 19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63 -derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 7 below. In some embodiments, a CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 19, 20, or 25, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 19, 20, or 25. In some embodiments, a CD19-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 21-23 and 26-28. In some embodiments, a CD19-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 21-23. In some embodiments, a CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 26-28. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of a CD 19 CAR comprises or consists of the one or more CDRs as described herein. In some embodiments, a CD19-specific scFv comprises CDRs defined by Kabat numbering. In some embodiments, a CD19-specific scFv comprises CDRs defined by Chothia numbering.

[0224] In some embodiments, a linker linking the VH and the VL portions of an scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO:24. In some embodiments, a Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO:30, which gives rise to a different FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:29. In certain of these embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:29.Table 7. Exemplary sequences of anti-CD19 scFv and components

[0225] In some embodiments, the extracellular binding domain of a CD 19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102: 15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)). In any of these embodiments, the extracellular binding domain of the CD 19 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0226] In some embodiments, the hinge domain of a CD 19 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:9. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 13.

[0227] In some embodiments, the transmembrane domain of a CD 19 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 15.

[0228] In some embodiments, the intracellular costimulatory domain of a CD 19 CAR comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In some embodiments, the 4- IBB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain. CD28 is another co-stimulatory molecule on T cells. In some embodiments, the CD28 costimulatory domain is human. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the intracellular costimulatory domain of the CD 19 CAR comprises a 4- IBB costimulatory domain and a CD28 costimulatory domain as described.

[0229] In some embodiments, the intracellular signaling domain of a CD 19 CAR comprises a CD3 zeta (Q signaling domain. CD3(^ associates with T cell receptors (TCRs) to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs). The CD3(^ signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In some embodiments, the CD3(^ signaling domain is human. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0230] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD 19 CAR, including, for example, a CD 19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO: 19 or SEQ ID NO:29, the CD8a hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described.

[0231] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD 19 CAR, including, for example, a CD19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO: 19 or SEQ ID NO:29, the IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 12, the CD28 transmembrane domain of SEQ ID NO: 15, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the CD 19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described.

[0232] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD 19 CAR, including, for example, a CD 19 CAR comprising the CD19-specific scFv having sequences set forth in SEQ ID NO: 19 or SEQ ID NO:29, the CD28 hinge domain of SEQ ID NO: 10, the CD28 transmembrane domain of SEQ ID NO: 15, the CD28 costimulatory domain of SEQ ID NO: 17, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described.

[0233] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD 19 CAR as set forth in SEQ ID NO:4 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 4 (see Table 8). The encoded CD 19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 5 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:5, with the following components: CD8a signal peptide, FMC63 scFv (VL-Whitlow linker-Vu), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain.

[0234] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a commercially available embodiment of CD 19 CAR. Non-limiting examples of commercially available embodiments of CD 19 CARs expressed and / or encoded by T cells include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.

[0235] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding tisagenlecleucel or portions thereof. Tisagenlecleucel comprises a CD 19 CAR with the following components: CD8a signal peptide, FMC63 scFv (VL-3XG4S linker-Vu), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in tisagenlecleucel are provided in Table 8, with annotations of the sequences provided in Table 9.

[0236] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding lisocabtagene maraleucel or portions thereof. Lisocabtagene maraleucel comprises a CD 19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (Vi -Whitlow linker-Vu), IgG4 hinge domain, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in lisocabtagene maraleucel are provided in Table 8, with annotations of the sequences provided in Table 10.

[0237] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding axicabtagene ciloleucel or portions thereof.Axicabtagene ciloleucel comprises a CD19 CAR with the following components: GMCSFR- a or CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker-Vu), CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3(^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in axicabtagene ciloleucel are provided in Table 8, with annotations of the sequences provided in Table 11.

[0238] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding brexucabtagene autoleucel or portions thereof. Brexucabtagene autoleucel comprises a CD19 CAR with the following components: GMCSFR- a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3(^ signaling domain.

[0239] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD 19 CAR as set forth in SEQ ID NO: 31, 33, or 35, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 31, 33, or 35. The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 32, 34, or 36, respectively, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 32, 34, or 36, respectively.Table 8. Exemplary sequences of CD19 CARsTable 9. Annotation of tisagenlecleucel CD19 CAR sequencesTable 10. Annotation of lisocabtagene maraleucel CD19 CAR sequencesTable 11. Annotation of axicabtagene ciloleucel CD19 CAR sequences

[0240] In some embodiments, a polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding CD19 CAR as set forth in SEQ ID NO: 31, 33, or 35, or at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 31, 33, or 35. The encoded CD 19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 32, 34, or 36, respectively, is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 32, 34, or 36, respectively.6. CD20 CAR

[0002] In some embodiments, the CAR is a CD20 CAR (“CD20-CAR”), and in these embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR. CD20 is an antigen found on the surface of B cells as early at the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkins disease, myeloma, and thymoma. In some embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain in tandem.

[0003] In some embodiments, the signal peptide of the CD20 CAR comprises a CD8a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NOR

[0004] In some embodiments, the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. The extracellular binding domain of the CD20 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0005] In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3,rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0006] In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from the Leul6 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leul6 connected by a linker. See Wu et al., Protein Engineering. 14(12): 1025-1033 (2001). In some embodiments, the linker is a 3xG4S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of different portions of the entire Leul6-derived scFv (also referred to as Leul6 scFv) and its different portions are provided in Table 11 below. In some embodiments, the CD20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:37, 38, or 42, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:37, 38, or 42. In some embodiments, the CD20- specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 39-41, 43 and 44. In some embodiments, the CD20-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 39-41. In some embodiments, the CD20-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 43-44. In any of these embodiments, the CD20-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD20 CAR comprises or consists of the one or more CDRs as described herein.Table 11. Exemplary sequences of anti-CD20 scFv and components

[0007] In some embodiments, the hinge domain of the CD20 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:9. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 13.

[0008] In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence that is at least 80% identical (e.g, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 15.

[0009] In some embodiments, the intracellular costimulatory domain of the CD20 CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 17.

[0010] In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0011] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37, the CD8a hinge domain of SEQ ID NO: 9, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0012] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37, the CD28 hinge domain of SEQ ID NO: 10, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0013] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37, the IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 12, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0014] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37, the CD8a hinge domain of SEQ ID NO:9, the CD28 transmembrane domain of SEQ ID NO: 15,the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0015] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37, the CD28 hinge domain of SEQ ID NO: 10, the CD28 transmembrane domain of SEQ ID NO: 15, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0016] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37, the IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 1, the CD28 transmembrane domain of SEQ ID NO: 15, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.7. CD22 CAR

[0017] In some embodiments, the CAR is a CD22 CAR (“CD22-CAR”), and in these embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR. CD22, which is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells. In some embodiments, the CD22 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain in tandem.

[0018] In some embodiments, the signal peptide of the CD22 CAR comprises a CD8a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NOR

[0019] In some embodiments, the extracellular binding domain of the CD22 CAR is specific to CD22, for example, human CD22. The extracellular binding domain of the CD22 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0020] In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific to CD22, including, for example, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0021] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from the m971 monoclonal antibody (m971), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of m971 connected by a linker. In some embodiments, the linker is a 3xG.rS linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971 -derived scFv (also referred to as m971 scFv) and its different portions are provided in Table 12 below. In some embodiments, the CD22-specific scFv comprises orconsists of an amino acid sequence set forth in SEQ ID NO:45, 46, or 50, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:45, 46, or 50. In some embodiments, the CD22- specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 47-49 and 51-53. In some embodiments, the CD22-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 47-49. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 51-53. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of the one or more CDRs as described herein.

[0022] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971-L7, which is an affinity matured variant of m971 with significantly improved CD22 binding affinity compared to the parental antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv derived from m971-L7 comprises the VH and the VL of m971-L7 connected by a 3xG4S linker. In other embodiments, the Whitlow linker may be used instead. In some embodiments, the amino acid sequences of the entire m971-L7-derived scFv (also referred to as m971-L7 scFv) and its different portions are provided in Table 12 below. In some embodiments, the CD22- specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 54, 55, or 59, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:54, 55, or 59. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 56-58 and 60-62. In some embodiments, the CD22- specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 56-58. In some embodiments, the CD22-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 60-62. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising asequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of the one or more CDRs as described herein.Table 12. Exemplary sequences of anti-CD22 scFv and components

[0023] In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise an scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of the cancer cells that express CD22 and kill the cancer cells. BL22 comprises a dsFv of an anti-CD22 antibody, RFB4, fused to a 38-kDa truncated form of Pseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11 : 1545-50 (2005)). HA22 (CAT8015, moxetumomab pasudotox) is a mutated, higher affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1): 607-17 (2005)). Suitable sequences of antigen binding domains of HA22 and BL22 specific to CD22 are disclosed in, for example, U.S. Patent Nos.7,541,034; 7,355,012; and 7,982,011, which are hereby incorporated by reference in their entirety.

[0024] In some embodiments, the hinge domain of the CD22 CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:9. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 13.

[0025] In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth inSEQ ID NO: 15 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 15.

[0026] In some embodiments, the intracellular costimulatory domain of the CD22 CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 17.

[0027] In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0028] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45 or SEQ ID NO: 54, the CD8a hinge domain of SEQ ID NO: 9, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0029] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45 or SEQ ID NO: 54, the CD28 hinge domain of SEQ ID NO: 10, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0030] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45 or SEQ ID NO: 54, the IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 12, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0031] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45 or SEQ ID NO: 54, the CD8a hinge domain of SEQ ID NO: 9, the CD28 transmembrane domain of SEQ ID NO: 15, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0032] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45 or SEQ ID NO: 54, the CD28 hinge domain of SEQ ID NO: 10, the CD28 transmembrane domain of SEQ ID NO: 15, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.

[0033] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45 or SEQ ID NO: 54, the IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 12, the CD28 transmembrane domain of SEQ ID NO: 15, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having asequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof.8. BCMA CAR

[0034] In some embodiments, the CAR is a BCMA CAR (“BCMA-CAR”), and in these embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a BCMA CAR. BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the BCMA CAR may comprise a signal peptide, an extracellular binding domain that specifically binds BCMA, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain in tandem.

[0035] In some embodiments, the signal peptide of the BCMA CAR comprises a CD8a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NOR

[0036] In some embodiments, the extracellular binding domain of the BCMA CAR is specific to BCMA, for example, human BCMA. The extracellular binding domain of theBCMA CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain.

[0037] In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv. In some embodiments, the extracellular binding domain of the BCMA CAR is derived from an antibody specific to BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, and ciltacabtagene. In any of these embodiments, the extracellular binding domain of the BCMA CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0038] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from Cl 1D5.3, a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. W02010 / 104949. The Cl lD5.3-derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of Cl 1D5.3 connected by the Whitlow linker, the amino acid sequences of which is provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:63, 64, or 68, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:63, 64, or 68. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67 and 69-71. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67. In some embodiments, the BCMA- specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 69-71. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.

[0039] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as describedin Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010 / 104949, the amino acid sequence of which is also provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:72, 73, or 77, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:72, 73, or 77. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 74-76 and 78-80. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 74-76. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 78-80. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.

[0040] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT Application Publication No. WO2012163805.

[0041] In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol. 11(1): 141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO2018 / 028647.

[0042] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11 (1) :283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. W02019 / 006072. The amino acid sequences of FHVH33 and its CDRs are provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:81 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:81. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 82-84. In any of these embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.

[0043] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No.11,026,975 B2, the amino acid sequence of which is provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 118, 119, or 123, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 118, 119, or 123. In some embodiments, the BCMA- specific extracellular binding domain may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 120-122 and 124-126. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 120-122. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 124-126. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of the one or more CDRs as described herein.

[0044] Additionally, CARs and binders directed to BCMA have been described in U.S. Application Publication Nos. 2020 / 0246381 Al and 2020 / 0339699 Al, the entire contents of each of which are incorporated by reference herein.Table 13. Exemplary sequences of anti-BCMA binder and components

[0045] In some embodiments, the hinge domain of the BCMA CAR comprises a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:9. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises orconsists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-Ch3 domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 13.

[0046] In some embodiments, the transmembrane domain of the BCMA CAR comprises a CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, for example, a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence that is at least 80% identical (e.g, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 15.

[0047] In some embodiments, the intracellular costimulatory domain of the BCMA CAR comprises a 4-1BB costimulatory domain, for example, a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, for example, a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 17.

[0048] In some embodiments, the intracellular signaling domain of the BCMA CAR comprises a CD3 zeta (Q signaling domain, for example, a human CD3(^ signaling domain. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0049] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, the CD8a hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of SEQ ID NO: 14, the 4-1BB costimulatory domain of SEQ ID NO: 16, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide) as described.

[0050] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains as described, the CD8a hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of SEQ ID NO: 14, the CD28 costimulatory domain of SEQ ID NO: 17, the CD3(^ signaling domain of SEQ ID NO: 18, and / or variants (i.e., having a sequence that is at least 80% identical, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence) thereof. In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described.

[0051] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a BCMA CAR as set forth in SEQ ID NO: 127 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 127 (see Table 14). The encoded BCMA CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 128 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 128, with thefollowing components: CD8a signal peptide, CT103A scFv (VL- Whitlow linker-Vu), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain.

[0052] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding a commercially available embodiment of BCMA CAR, including, for example, idecabtagene vicleucel (ide-cel, also called bb2121). In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding idecabtagene vicleucel or portions thereof. Idecabtagene vicleucel comprises a BCMA CAR with the following components: the BB2121 binder, CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain.Table 14. Exemplary sequences of BCMA CARsC. Methods of Treatment with Immunosuppression-Resistant T Cells

[0241] As is described in further detail herein, provided herein are methods for treating a patient with a disease, disorder, or condition through administration of immunosuppression-resistant cells, particularly immunosuppression-resistant T cells. In some embodiments, immunosuppression-resistant T cells express a chimeric antigen receptor (CAR). In some embodiments, immunosuppression-resistant cells, particularly immunosuppression-resistant T cells are also engineered to be hypoimmunogenic. As will be appreciated, for all the multiple embodiments described herein related to the timing and / or combinations of therapies, administration of the cells is accomplished by a method or route which results in at least partial localization of the introduced cells at a desired site. Cells can be infused, implanted, or transplanted directly to the desired site, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.

[0242] Provided herein are methods for treating a patient with a disease, disorder, or condition of the present disclosure, which includes administration of a population of immunosuppression-resistant cells (e.g., primary T cells) to a subject, e.g., a human patient. For instance, a population of immunosuppression-resistant primary T cells such as, but limited to, CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Thl cells, Th2 cells, Th9 cells, Thl7 cells, T-follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tern) cells, effector memory T cells that express CD45RA (TEMRA cells), tissue-resident memory (Trm) cells, virtual memory T cells, innate memory T cells, memory stem cell (Tsc), yA T cells, and any other subtype of T cell is administered to a patient to treat a disease, disorder, or condition of the present disclosure. In some embodiments, a population of immunosuppression-resistant cells to be administered to a subject is also engineered to be hypoimmunogenic.

[0243] An engineered T cell comprising reduced expression of FKBP12 relative to a comparable or wild-type T cell can be used, inter alia, to treat antibody -mediated rejection (AMR) and / or to reduce a level of graft-directed antibodies in a subject.

[0244] A subject to be treated with methods described herein can be e.g., a patient having, at risk of having, or diagnosed as having antibody-mediated rejection. A subject to be treated with methods described herein can be e.g., a patient with one or more graft-directed antibodies.

[0245] In some embodiments, a method of treating antibody -mediated rejection in a subject comprises administering to a subject a composition according to the present disclosure. In some embodiments, a subject has one or more graft-directed antibodies. In some embodiments, administration of a composition according to the present disclosure reduces a level of graft-directed antibody, as compared to a subject who has not been administered the composition, or as compared to the same subject prior to administration of the composition. In some embodiments, a reduction in the level of the graft-directed antibody prevents graft failure.1. Dosing and Formulations

[0246] Any therapeutically effective amount of cells described herein can be included in a pharmaceutical composition of the present disclosure, depending on the indication beingtreated. Non-limiting examples of the cells include primary T cells. In some embodiments, a pharmaceutical composition includes at least about IxlO2, 5xl02, IxlO3, 5xl03, IxlO4, 5xl04, IxlO5, 5xl05, IxlO6, 5xl06, IxlO7, 5xl07, IxlO8, 5xl08, IxlO9, 5xl09, IxlO10, or 5xlO10cells. In some embodiments, the pharmaceutical composition includes up to about IxlO2, 5xl02, IxlO3, 5xl03, IxlO4, 5xl04, IxlO5, 5xl05, IxlO6, 5xl06, IxlO7, 5xl07, IxlO8, 5xl08, IxlO9, 5xl09, IxlO10, or 5xlO10cells. In some embodiments, the pharmaceutical composition includes up to about 6.0 x 108cells. In some embodiments, the pharmaceutical composition includes up to about 8.0 x 108cells. In some embodiments, the pharmaceutical composition includes at least 30xl06cells. In some embodiments, the pharmaceutical composition includes approximately 30xl06cells. In some embodiments, the pharmaceutical composition includes at least 60xl06cells. In some embodiments, the pharmaceutical composition includes approximately 60xl06cells. In some embodiments, the pharmaceutical composition includes at least 90xl06cells. In some embodiments, the pharmaceutical composition includes approximately 90xl06cells. In some embodiments, the pharmaceutical composition includes at least 120xl06cells. In some embodiments, the pharmaceutical composition includes approximately 120xl06cells. In some embodiments, the pharmaceutical composition includes at least 200xl06cells. In some embodiments, the pharmaceutical composition includes approximately 200xl06cells. In some embodiments, the pharmaceutical composition includes at least about IxlO2to 5xl02, 5xl02to IxlO3, IxlO3to 5xl03, 5xl03to IxlO4, IxlO4to 5xl04, 5xl04to IxlO5, IxlO5to 5xl05, 5xl05to IxlO6, IxlO6to 5xl06, 5xl06to IxlO7, IxlO7to 5xl07, 5xl07to IxlO8, IxlO8to 5xl08, 5xl08to IxlO9, IxlO9to 5xl09, 5xl09to IxlO10, or IxlO10to 5xlO10cells. In some embodiments, the pharmaceutical composition includes from about l.OxlO6to about 2.5xl08cells. In some embodiments, the pharmaceutical composition includes from about 2.0xl06to about 2.0xl08cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises from about 30xl06cells to about 200xl06cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises from about 20xl06cells to about 400xl06cells.

[0247] In some embodiments, a pharmaceutical composition includes at least about IxlO2, 5xl02, IxlO3, 5xl03, IxlO4, 5xl04, IxlO5, 5xl05, IxlO6, 5xl06, IxlO7, 5xl07, IxlO8, 5xl08, IxlO9, 5xl09, IxlO10, or 5xlO10CAR+ cells. In some embodiments, the pharmaceutical composition includes up to about IxlO2, 5xl02, IxlO3, 5xl03, IxlO4, 5xl04, IxlO5, 5xl05, IxlO6, 5xl06, IxlO7, 5xl07, IxlO8, 5xl08, IxlO9, 5xl09, IxlO10, or 5xlO10CAR+ cells. In some embodiments, the pharmaceutical composition includes up to about 6.0xl08CAR+ cells. In some embodiments, the pharmaceutical composition includes up to about 8.0xl08CAR+ cells. In some embodiments, the pharmaceutical composition includes at least 30xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes approximately 30xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes at least 60xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes approximately 60xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes at least 120xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes approximately 120xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes at least 200xl06CAR+ cells. In some embodiments, the pharmaceutical composition includes approximately 200x106CAR+ cells. In some embodiments, the pharmaceutical composition includes at least about 1 x 102to 5 x 102, 5 x 102to 1 x 103, 1 x 103to 5 x 103, 5 x 103to 1 x 104, 1 x 104to 5 x 104, 5 x 104to 1 x 105, 1 x 105to 5 x 105, 5 x 105to 1 x 106, 1 x 106to 5 x 106, 5 x 106to 1 x 107, 1 x 107to 5 x 107, 5 x 107to 1 x 108, 1 x 108to 5 x 108, 5 x 108to 1 x 109, 1 x 109to 5 x 109, 5 x 109to 1 x IO10, or 1 x IO10to 5 x IO10CAR+ cells. In some embodiments, the pharmaceutical composition includes from about 1.0 x 106to about 2.5 x 108CAR+ cells. In some embodiments, the pharmaceutical composition includes from about 2.0 x 106to about 2.0 x 108CAR+ cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises from about 30xl06CAR+ cells to about 200xl06CAR+ cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises about 5xl06CAR+ cells to at least about 400xl06CAR+ cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises about 20xl06CAR+ cells to about 400xl06CAR+ cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises at least about 30xl06CAR+ cells, at least about 60xl06CAR+ cells, at least about 120xl06CAR+ cells, or at least about 200xl06CAR+ cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises no more than about 200xl06CAR+ cells. In some embodiments, a pharmaceutical composition of the present disclosure comprises about 40 x 106, 41 x 106, 42 x 106, 43 x 106, 44 x 106, 45 x 106, 46 x 106, 47 x 106, 48 x 106, 49 x 106, 50 x 106, 51 x 106, 52 x 106, 53 x 106, 54 x 106, 55 x 106, 56 x 106, 57 x 106, 58 x 106, 59 x 106, 60 x 106, 61 x 106, 62 x 106, 63 x 106, 64 x 106, 65 x 106, 66 x 106, 67 x 106, 68 x 106, 69 x 106, 70 x 106, 71 x 106, 72 x 106, 73 x 106, 74 x 106, 75 x 106, 76 x 106, 77 x 106, 78 x 106, 79 x 106, 80 x 106, 81 x 106, 82 x 106, 83 x 106, 84 x 106, 85 x 106, 86 x 106, 87 x 106, 88 x 106, 89 x 106, 90 x 106, 91 x 106, 92 x 106, 93 x 106, 94 x 106, 95 x 106, 96X 106, 97 X 106, 98 X 106, 99 x 106, 100 x 106, 101 x 106, 102 x 106, 103 x 106, 104 x 106, 105 x 106, 106 x 106, 107 x 106, 108 x 106, 109 x 106, 110 x 106, 111 x 106, 112 x 106, 113 x 106, 114 x 106, 115 x 106, 116 x 106, 117 x 106, 118 x 106, 119 x 106, 120 x 106, 121 x 106, 122 x 106, 123 x 106, 124 x 106, 125 x 106, 126 x 106, 127 x 106, 128 x 106, 129 x 106, 130 x 106, 131 x 106, 132 x 106, 133 x 106, 134 x 106, 135 x 106, 136 x 106, 137 x 106, 138 x 106, 139 x 106, 140 x 106, 141 x 106, 142 x 106, 143 x 106, 144 x 106, 145 x 106, 146 x 106, 147 x 106, 148 x 106, 149 x 106, 150 x 106, 151 x 106, 152 x 106, 153 x 106, 154 x 106, 155 x 106, 156 x 106, 157 x 106, 158 x 106, 159 x 106, 160 x 106, 161 x 106, 162 x 106, 163 x 106, 164 x 106, 165 x 106, 166 x 106, 167 x 106, 168 x 106, 169 x 106, 170 x 106, 171 x 106, 172 x 106, 173 x 106, 174 x 106, 175 x 106, 176 x 106, 177 x 106, 178 x 106, 179 x 106, 180 x 106, 181 x 106, 182 x 106, 183 x 106, 184 x 106, 185 x 106, 186 x 106, 187 x 106, 188 x 106, 189 x 106, 190 x 106, 191 x 106, 192 x 106, 193 x 106, 194 x 106, 195 x 106, 196 x 106, 197 x 106, 198 x 106, 199 x 106, 200 x 106, 201 x 106, 202 x 106, 203 x 106, 204 x 106, 205 x 106, 206 x 106, 207 x 106, 208 x 106, 209 x 106, 210 x 106, 211 x 106, 212 x 106, 213 x 106, 214 x 106, 215 x 106, 216 x 106, 217 x 106, 218 x 106, 219 x 106, 220 x 106, 221 x 106, 222 x 106, 223 x 106, 224 x 106, 225 x 106, 226 x 106, 227 x 106, 228 x 106, 229 x 106, 230 x 106, 231 x 106, 232 x 106, 233 x 106, 234 x 106, 235 x 106, 236 x 106, 237 x 106, 238 x 106, 239 x 106, 240 x 106, 241 x 106, 242 x 106, 243 x 106, 244 x 106, 245 x 106, 246 x 106, 247 x 106, 248 x 106, 249 x 106, or 250 x 106CAR+ cells.

[0248] In some embodiments, a pharmaceutical composition of the present disclosure comprises immunosuppression-resistant CAR+ cells as described herein. In some embodiments, immunosuppression-resistant CAR+ cells comprise one or more modifications which inactivate or disrupt FKBP1A. In some embodiments, immunosuppression-resistant CAR+ cells demonstrated reduced expression of FKBP12 relative to a comparable T cell or a wild type T cell.

[0249] In some embodiments, a pharmaceutical composition of the present disclosure comprises immunosuppression-resistant CAR+ cells which are further engineered (e.g., fully edited) to be hypoimmunogenic. In some embodiments, fully edited hypoimmunogenic CAR+ cells comprise one or more modifications that: (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules; and / or (ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules; and / or (iii) one or more T-cellreceptor (TCR) molecules and / or one or more molecules that regulate expression of the one or more TCR molecules, (b) increase expression of a tolerogenic factor encoded by a first exogenous polynucleotide, wherein the increased expression of (b) is relative to a comparable T cell or wild-type T cell that does not comprise the modification, and (c) express a chimeric antigen receptor (CAR) encoded by a second exogenous polynucleotide.

[0250] In some embodiments, a pharmaceutical composition has a volume of at least 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 mL. In exemplary embodiments, a pharmaceutical composition has a volume of up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 mL. In exemplary embodiments, a pharmaceutical composition has a volume of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 mL. In some embodiments, a pharmaceutical composition has a volume of from about 1-50 mL, 50-100 mL, 100-150 mL, 150-200 mL, 200-250 mL, 250-300 mL, 300-350 mL, 350-400 mL, 400-450 mL, or 450-500 mL. In some embodiments, a pharmaceutical composition has a volume of from about 1-50 mL, 50-100 mL, 100-150 mL, 150-200 mL, 200-250 mL, 250-300 mL, 300-350 mL, 350-400 mL, 400- 450 mL, or 450-500 mL. In some embodiments, a pharmaceutical composition has a volume of from about 1-10 mL, 10-20 mL, 20-30 mL, 30-40 mL, 40-50 mL, 50-60 mL, 60-70 mL, 70-80 mL, 70-80 mL, 80-90 mL, or 90-100 mL. In some embodiments, a pharmaceutical composition has a volume that ranges from about 1.25mL to about lOOmL. In some embodiments, a pharmaceutical composition has a volume that ranges from about 5 mL to about 80 mL. In some embodiments, a pharmaceutical composition has a volume that ranges from about 10 mL to about 70 mL. In some embodiments, a pharmaceutical composition has a volume that ranges from about 10 mL to about 50 mL. In some embodiments, a pharmaceutical composition has a volume of at least about 7.5 mL, at least about 15 mL, at least about 30 mL, or at least about 50 mL.

[0251] In some embodiments, a specific amount / dosage regimen will vary depending on the weight, gender, age and health of a subject; the formulation, the biochemical nature, bioactivity, bioavailability and the side effects of the cells and the number and identity of the cells in the complete therapeutic regimen.

[0252] In some embodiments, a therapeutically effective dose or a clinically effective dose of the pharmaceutical composition includes about l.OxlO5to about 2.5xl08cells at a volume of about 10 mL to 50 mL and the pharmaceutical composition is administered as a single therapeutically effective dose or clinically effective dose. In some cases, the therapeutically effective dose or clinically effective dose includes about l.OxlO5to about 2.5xl08primary T cells described herein at a volume of about 10 mL to 50 mL. In some embodiments, a therapeutically effective dose or clinically effective dose includes about l.OxlO5to about 2.5xl08primary T cells as disclosed herein at a volume of about 10 mL to 50 mL.

[0253] In some embodiments, a therapeutically effective dose or a clinically effective dose of the pharmaceutical composition comprises a concentration of engineered immunosuppression-resistant T cells of at least 1.25xl06cells / mL. In some embodiments, a therapeutically effective dose or a clinically effective dose of the pharmaceutical composition comprises a concentration of engineered immunosuppression-resistant T cells of at least 4.0xl06cells / mL. In some embodiments, a therapeutically effective dose or a clinically effective dose of the pharmaceutical composition comprises a concentration of engineered immunosuppression-resistant T cells of at least 1.2xl06CAR+ cells / mL. In some embodiments, a therapeutically effective dose or a clinically effective dose of the pharmaceutical composition comprises a concentration of engineered immunosuppressionresistant T cells of at least 4.0xl06CAR+ cells / mL.

[0254] In some embodiments, the pharmaceutical composition is administered as a single therapeutically effective dose or clinically effective dose of from about l.OxlO5to about l.OxlO7cells (such as primary T cells) per kg body weight for subjects 50 kg or less. In some embodiments, the pharmaceutical composition is administered as a single therapeutically effective dose or clinically effective dose of from about 0.5xl05to about l.OxlO7, about l.OxlO5to about l.OxlO7, about l.OxlO5to about l.OxlO7, about 5.0xl05to about IxlO7, about l.OxlO6to about IxlO7, about 5.0xl06to about l.OxlO7, about l.OxlO5to about 5.0xl06, about l.OxlO5to about l.OxlO6, about l.OxlO5to about 5.0xl05, about l.OxlO5to about 5.0xl06, about 2.0xl05to about 5.0xl06, about 3.0xl05to about 5.0xl06, about 4.0xl05to about 5.0xl06, about 5.0xl05to about 5.0xl06, about 6.0xl05to about 5.0xl06, about 7.0xl05to about 5.0xl06, about 8.0xl05to about 5.0xl06, or about 9.0xl05to about 5.0xl06cells per kg body weight for subjects 50 kg or less. In some embodiments, the therapeutically effective dose or clinically effective dose is 0.5 x 105, 0.6 x 105, 0.7 x 105, 0.8X IO5, 0.9 x lO5, 1.0 x lO5, 1.1 x lO5, 1.2 x lO5, 1.3 x lO5, 1.4 x lO5, 1.5 x lO5, 1.6 x lO5, 1.7 x IO5, 1.8 x IO5, 1.9 x IO5, 2.0 x IO5, 2.1 x IO5, 2.2 x IO5, 2.3 x IO5, 2.4 x IO5, 2.5 x IO5, 2.6 xIO5, 2.7 x IO5, 2.8 x IO5, 2.9 x IO5, 3.0 x IO5, 3.1 x IO5, 3.2 x IO5, 3.3 x IO5, 3.4 x IO5, 3.5 xIO5, 3.6 x IO5, 3.7 x IO5, 3.8 x IO5, 3.9 x IO5, 4.0 x IO5, 4.1 x IO5, 4.2 x IO5, 4.3 x IO5, 4.4 x105, 4.5 x IO5, 4.6 x IO5, 4.7 x IO5, 4.8 x IO5, 4.9 x IO5, 5.0 x IO5, 0.5 x 106, 0.6 x 106, 0.7 x106, 0.8 x 106, 0.9 x 106, 1.0 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x106, 1.7 x 106, 1.8 x 106, 1.9 x 106, 2.0 x 106, 2.1 x 106, 2.2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3.0 x 106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x 106, 3.9 x 106, 4.0 x 106, 4.1 x 106, 4.2 x 106, 4.3 x106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x 106, 4.8 x 106, 4.9 x 106, 5.0 x 106, 5.1 x 106, 5.2 x106, 5.3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x 106, 5.7 x 106, 5.8 x 106, 5.9 x 106, 6.0 x 106, 6.1 x106, 6.2 x 106, 6.3 x 106, 6.4 x 106, 6.5 x 106, 6.6 x 106, 6.7 x 106, 6.8 x 106, 6.9 x 106, 7.0 x106, 7.1 x 106, 7.2 x 106, 7.3 x 106, 7.4 x 106, 7.5 x 106, 7.6 x 106, 7.7 x 106, 7.8 x 106, 7.9 x106, 8.0 x 106, 8.1 x 106, 8.2 x 106, 8.3 x 106, 8.4 x 106, 8.5 x 106, 8.6 x 106, 8.7 x 106, 8.8 x106, 8.9 x 106, 9.0 x 106, 9.1 x 106, 9.2 x 106, 9.3 x 106, 9.4 x 106, 9.5 x 106, 9.6 x 106, 9.7 x106, 9.8 x 106, 9.9 x 106, 0.5 x 107, 0.6 x 107, 0.7 x 107, 0.8 x 107, 0.9 x 107, or 1.0 x 107cells per kg body weight for subjects 50 kg or less. In some embodiments, a therapeutically effective dose or clinically effective dose is from about 0.2 x 106to about 5.0 x 106cells per kg body weight for subjects 50 kg or less. In certain embodiments, a therapeutically effective dose or clinically effective dose is at a range that is lower than from about 0.2 x 106to about 5.0 x 106cells per kg body weight for subjects 50 kg or less. In some embodiments, a single therapeutically effective dose or clinically effective dose is at a volume of about 10 mL to 50 mL. In some embodiments, a therapeutically effective dose or clinically effective dose is administered intravenously. In some embodiments, a therapeutically effective dose or clinically effective dose is administered to the nervous system of a subject. In some embodiments, the nervous system is the central nervous system (CNS). In some embodiments, the nervous system is the peripheral nervous system (PNS). In some embodiments, a therapeutically effective dose or clinically effective dose is administered intrathecally.

[0255] In exemplary embodiments, cells are administered in a single therapeutically effective dose of from about l.OxlO6to about 5.0xl08cells (such as primary T cells) for subjects above 50 kg. In some embodiments, a pharmaceutical composition is administered as a single therapeutically effective dose or clinically effective dose of from about 0.5xl06toabout l.OxlO9, about l.OxlO6to about l.OxlO9, about l.OxlO6to about l.OxlO9, about 5.0xl06to about l.OxlO9, about l.OxlO7to about l.OxlO9, about 5.0xl07to about l.OxlO9, about l.OxlO6to about 5.0xl07, about l.OxlO6to about l.OxlO7, about l.OxlO6to about 5.0xl07, about l.OxlO7to about 5.0xl08, about 2.0xl07to about 5.0xl08, about 3.0xl07to about 5.0xl08, about 4.0xl07to about 5.0xl08, about 5.0xl07to about 5.0xl08, about 6.0xl07to about 5.0xl08, about 7.0xl07to about 5.0xl08, about 8.0xl07to about 5.0xl08, or about 9.0xl07to about 5.0xl08cells per kg body weight for subjects 50 kg or less. In some embodiments, a therapeutically effective dose or clinically effective dose is 1.0 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, 2.0 x106, 2.1 x 106, 2.2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x106, 3.0 x 106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x106, 3.9 x 106, 4.0 x 106, 4.1 x 106, 4.2 x 106, 4.3 x 106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x106, 4.8 x 106, 4.9 x 106, 5.0 x 106, 5.1 x 106, 5.2 x 106, 5.3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x106, 5.7 x 106, 5.8 x 106, 5.9 x 106, 6.0 x 106, 6.1 x 106, 6.2 x 106, 6.3 x 106, 6.4 x 106, 6.5 x106, 6.6 x 106, 6.7 x 106, 6.8 x 106, 6.9 x 106, 7.0 x 106, 7.1 x 106, 7.2 x 106, 7.3 x 106, 7.4 x106, 7.5 x 106, 7.6 x 106, 7.7 x 106, 7.8 x 106, 7.9 x 106, 8.0 x 106, 8.1 x 106, 8.2 x 106, 8.3 x106, 8.4 x 106, 8.5 x 106, 8.6 x 106, 8.7 x 106, 8.8 x 106, 8.9 x 106, 9.0 x 106, 9.1 x 106, 9.2 x106, 9.3 x 106, 9.4 x 106, 9.5 x 106, 9.6 x 106, 9.7 x 106, 9.8 x 106, 9.9 x 106, 1.0 x 107, 1.1 x107, 1.2 x 107, 1.3 x 107, 1.4 x 107, 1.5 x 107, 1.6 x 107, 1.7 x 107, 1.8 x 107, 1.9 x 107, 2.0 x107, 2.1 x 107, 2.2 x 107, 2.3 x 107, 2.4 x 107, 2.5 x 107, 2.6 x 107, 2.7 x 107, 2.8 x 107, 2.9 x107, 3.0 x 107, 3.1 x 107, 3.2 x 107, 3.3 x 107, 3.4 x 107, 3.5 x 107, 3.6 x 107, 3.7 x 107, 3.8 x107, 3.9 x 107, 4.0 x 107, 4.1 x 107, 4.2 x 107, 4.3 x 107, 4.4 x 107, 4.5 x 107, 4.6 x 107, 4.7 x107, 4.8 x 107, 4.9 x 107, 5.0 x 107, 5.1 x 107, 5.2 x 107, 5.3 x 107, 5.4 x 107, 5.5 x 107, 5.6 x107, 5.7 x 107, 5.8 x 107, 5.9 x 107, 6.0 x 107, 6.1 x 107, 6.2 x 107, 6.3 x 107, 6.4 x 107, 6.5 x107, 6.6 x 107, 6.7 x 107, 6.8 x 107, 6.9 x 107, 7.0 x 107, 7.1 x 107, 7.2 x 107, 7.3 x 107, 7.4 x107, 7.5 x 107, 7.6 x 107, 7.7 x 107, 7.8 x 107, 7.9 x 107, 8.0 x 107, 8.1 x 107, 8.2 x 107, 8.3 x107, 8.4 x 107, 8.5 x 107, 8.6 x 107, 8.7 x 107, 8.8 x 107, 8.9 x 107, 9.0 x 107, 9.1 x 107, 9.2 x107, 9.3 x 107, 9.4 x 107, 9.5 x 107, 9.6 x 107, 9.7 x 107, 9.8 x 107, 9.9 x 107, 1.0 x 108, 1.1 x108, 1.2 x 108, 1.3 x 108, 1.4 x 108, 1.5 x 108, 1.6 x 108, 1.7 x 108, 1.8 x 108, 1.9 x 108, 2.0 x108, 2.1 x 108, 2.2 x 108, 2.3 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.7 x 108, 2.8 x 108, 2.9 x108, 3.0 x 108, 3.1 x 108, 3.2 x 108, 3.3 x 108, 3.4 x 108, 3.5 x 108, 3.6 x 108, 3.7 x 108, 3.8 x108, 3.9 x 108, 4.0 x 108, 4.1 x 108, 4.2 x 108, 4.3 x 108, 4.4 x 108, 4.5 x 108, 4.6 x 108, 4.7 x108, 4.8 x 108, 4.9 x 108, or 5.0 x 108cells per kg body weight for subjects 50 kg or less. In certain embodiments, cells are administered in a single therapeutically effective dose orclinically effective dose of about 1.0 x 107to about 2.5 x 108cells for subjects above 50 kg. In some embodiments, cells are administered in a single therapeutically effective dose or clinically effective dose of a range that is less than about 1.0 x 107to about 2.5 x 108cells for subjects above 50 kg. In some embodiments, cells are administered in a single therapeutically effective dose or clinically effective dose of a range that is higher than about 1.0 x 107to about 2.5 x 108cells for subjects above 50 kg. In some embodiments, a dose is administered intravenously. In some embodiments, a therapeutically effective dose or clinically effective dose is administered to the nervous system of a subject. In some embodiments, the nervous system is the central nervous system (CNS). In some embodiments, the nervous system is the peripheral nervous system (PNS). In some embodiments, a therapeutically effective dose or clinically effective dose is administered intrathecally. In some embodiments, a single therapeutically effective dose or clinically effective dose is at a volume of about 10 mL to 50 mL. In some embodiments, a therapeutically effective dose or clinically effective dose is administered intravenously. In some embodiments, a therapeutically effective dose or clinically effective dose is administered to the nervous system of a subject. In some embodiments, the nervous system is the central nervous system (CNS). In some embodiments, the nervous system is the peripheral nervous system (PNS). In some embodiments, a therapeutically effective dose or clinically effective dose is administered intrathecally.

[0256] In exemplary embodiments, the therapeutically effective dose or clinically effective dose is administered intravenously at a rate of about 1 to 50 mL per minute, 1 to 40 mL per minute, 1 to 30 mL per minute, 1 to 20 mL per minute, 10 to 20 mL per minute, 10 to 30 mL per minute, 10 to 40 mL per minute, 10 to 50 mL per minute, 20 to 50 mL per minute, 30 to 50 mL per minute, 40 to 50 mL per minute. In numerous embodiments, a pharmaceutical composition is stored in one or more infusion bags for intravenous administration. In some embodiments, a dose is administered completely in no more than 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 240 minutes, or 300 minutes.

[0257] In some embodiments, a single therapeutically effective dose or clinically effective dose of the pharmaceutical composition is present in a single infusion bag. In other embodiments, a single therapeutically effective dose or clinically effective dose of a pharmaceutical composition is divided into 2, 3, 4 or 5 separate infusion bags.

[0258] In some embodiments, cells described herein are administered in a plurality of doses such as 2, 3, 4, 5, 6 or more doses, wherein the plurality of doses together constitute a therapeutically effective dose or clinically effective dose regimen. In some embodiments, each dose of a plurality of doses is administered to the subject ranging from 1 to 24 hours apart. In some instances, a subsequent dose is administered from about 1 hour to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or about 24 hours) after an initial or preceding dose. In some embodiments, each dose of a plurality of doses is administered to the subject ranging from about 1 day to 28 days apart. In some instances, a subsequent dose is administered from about 1 day to about 28 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or about 28 days) after an initial or preceding dose. In certain embodiments, each dose of a plurality of doses is administered to the subject ranging from 1 week to about 6 weeks apart. In certain instances, a subsequent dose is administered from about 1 week to about 6 weeks (e.g., about 1, 2, 3, 4, 5, or 6 weeks) after an initial or preceding dose. In several embodiments, each dose of a plurality of doses is administered to the subject ranging from about 1 month to about 12 months apart. In several instances, a subsequent dose is administered from about 1 month to about 12 months (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) after an initial or preceding dose.

[0259] In some embodiments, a subject is administered a first dosage regimen at a first time point, and then subsequently administered a second dosage regimen at a second time point. In some embodiments, the first dosage regimen is the same as the second dosage regimen. In other embodiments, the first dosage regimen is different than the second dosage regimen. In some instances, the number of cells in the first dosage regimen and the second dosage regimen are the same. In some instances, the number of cells in the first dosage regimen and the second dosage regimen are different. In some cases, the number of doses of the first dosage regimen and the second dosage regimen are the same. In some cases, the number of doses of the first dosage regimen and the second dosage regimen are different.2. Pharmaceutical Compositions

[0260] For therapeutic application, cells prepared according to the disclosed methods can typically be supplied in the form of a pharmaceutical composition comprising an isotonic excipient and are prepared under conditions that are sufficiently sterile for humanadministration. For general principles in medicinal formulation of cell compositions, see "Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy," by Morstyn & Sheridan eds, Cambridge University Press, 1996; and "Hematopoietic Stem Cell Therapy," E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The cells can be packaged in a device or container suitable for distribution or clinical use.

[0261] In some embodiments, pharmaceutical compositions of the present disclosure comprise a population of engineered immunosuppression-resistant T cells comprising reduced expression of FKBP12 relative to a comparable T cell or wild-type T cell. In some embodiments, immunosuppression-resistant T cells are hypoimmunogenic, further comprising: (a) reduced expression of Beta-2-Microglobulin (B2M), Class II Transactivator (CIITA), and / or T cell receptor alpha (TRAC) relative to a comparable or wild type T cell, (b) increased expression of CD47 encoded by a first exogenous polynucleotide relative to the control T cell, and (c) expression of a chimeric antigen receptor (CAR) encoded by a second exogenous polynucleotide, wherein the population of engineered immunosuppressionresistant hypoimmunogenic T cells are allogeneic to the subject receiving the pharmaceutical composition. a. Pharmaceutically acceptable carriers

[0262] In some embodiments, a pharmaceutical composition provided herein further include a pharmaceutically acceptable carrier. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); salts such as sodium chloride; and / or non-ionic surfactants such as polysorbates (TWEEN™), poloxamers (PLURONICS™) or polyethyleneglycol (PEG). In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable buffer (e.g., neutral buffer saline or phosphate buffered saline).

[0263] In some embodiments, a pharmaceutical composition includes one or more electrolyte base solutions selected from the group consisting of lactated CryoStor®, Ringer's solution, PlasmaLyte-A™, Iscove's Modified Dulbecco's Medium, Normosol-R™, Veen-D™, Polysal® and Hank's Balanced Salt Solution (containing no phenol red). These base solutions closely approximate the composition of extracellular mammalian physiological fluids.

[0264] In some embodiments, a pharmaceutical composition includes one or more cryoprotective agents selected from the group consisting of arabinogalactan, glycerol, polyvinylpyrrolidone (PVP), dextrose, dextran, trehalose, sucrose, raffinose, hydroxyethyl starch (HES), propylene glycol, human serum albumin (HSA), and dimethylsulfoxide (DMSO). In some embodiments, the pharmaceutically acceptable buffer is neutral buffer saline or phosphate buffered saline. In some embodiments, pharmaceutical compositions provided herein include one or more of CryoStor® CSB, Plasma-Lyte-A™, HSA, DMSO, and trehalose.

[0265] CryoStor® is an intracellular-like optimized solution containing osmotic / oncotic agents, free radical scavengers, and energy sources to minimize apoptosis, minimize ischemia / reperfusion injury and maximize the post-thaw recovery of the greatest numbers of viable, functional cells. CryoStor® is serum- and protein-free, and non- immunogenic. CryoStor® is cGMP -manufactured from raw materials of USP grade or higher. CryoStor® is a family of solutions pre-formulated with 0%, 2%, 5% or 10% DMSO. CryoStor® CSB is a DMSO-free version of CryoStor®. In some embodiments, a pharmaceutical composition includes a base solution of CryoStor® CSB at a concentration of about 0-100%, 5-95%, 10-90%, 15-85%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70- 80%, 25-75%, 30-70%, 35-65%, 40-60%, or 45-55% w / w. In some embodiments, a pharmaceutical composition includes a base solution of CryoStor® CSB at a concentration of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% w / w.

[0266] PlasmaLyte-A™ is a non-polymeric plasma expander and contains essential salts and nutrients similar to those found in culture medium but does not contain additional constituents found in tissue culture medium which are not approved for human infusion, e.g., phenol red, or are unavailable in U.S.P. grade. PlasmaLyte-A™ contains about 140 mEq / literIll of sodium (Na), about 5 mEq / liter of potassium (K), about 3 mEq / liter of magnesium (Mg), about 98 mEq / liter of chloride (Cl), about 27 mEq / liter of acetate, and about 23 mEq / liter of gluconate. (PlasmaLyte-A™ is commercially available from Baxter, Hyland Division, Glendale Calif., product No. 2B2543). In some embodiments, a pharmaceutical composition includes a base solution of PlasmaLyte-A™ at a concentration of about 0-100%, 5-95%, 10- 90%, 15-85%, 15-80%, 15-75%, 15-70%, 15-65%, 15-60%, 15-55%, 15-50%, 15-45%, 15- 40%, 15-35%, 15-30%, 15-25%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20- 50%, 20-45%, 20-40%, 20-35%, 20-30%, 25-75%, 30-70%, 35-65%, 40-60%, or 45-55% w / w. In some embodiments, the pharmaceutical composition includes a base solution of PlasmaLyte-A™ at a concentration of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% w / w.

[0267] In some embodiments, a pharmaceutical composition includes a DMSO concentration (v / v) of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25%. In some embodiments, a pharmaceutical composition includes a DMSO concentration (v / v) of about 7.5%.

[0268] In some embodiments, a pharmaceutical composition includes human serum albumin (HSA) at a concentration of about 0-10%, 0.3-9.3%, 0.3-8.3%, 0.3-7.3%, 0.3-6.3%, 0.3-5.3%, 0.3-4.3%, 0.3-3.3%, 0.3-2.3%, 0.3-1.3%, 0.6-8.3%, 0.9-7.3%, 1.2-6.3%, 1.5-5.3%, 1.8-4.3%, or 2.1-3.3% w / v. In some embodiments, a pharmaceutical composition comprises human serum albumin (HSA) at a concentration (w / v) of at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25%. In some embodiments, a pharmaceutical composition includes HSA at a concentration of about 0%, 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, 1.8%, 2.1%, 2.4%, 2.7%, 3.0%, 3.3%, 3.6%, 3.9%, 4.3%, 4.6%, 4.9%, 5.3%,5.6%, 5.9%, 6.3%, 6.6%, 6.9%, 7.3%, 7.6%, 7.9%, 8.3%, 8.6%, 8.9%, 9.3%, 9.6%, 9.9%, or 10% w / v. In some embodiments, a pharmaceutical composition comprises HSA at a concentration (w / v) of about 0.3%.

[0269] In some embodiments, a pharmaceutical composition includes dimethyl sulfoxide (DMSO) at a concentration of about 0-10%, 0.5-9.5%, 1-9%, 1.5-8.5%, 2-8%, 3- 8%, 4-8%, 5-8%, 6-8%, 7-8%, 2.5-7.5%, 3-7%, 3.5-6.5%, 4-6%, or 4.5-5.5% v / v. In some embodiments, a pharmaceutical composition includes HSA at a concentration of about 0%, 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10.0% v / v.

[0270] In some embodiments, a pharmaceutical composition includes dimethyl sulfoxide (DMSO) at a concentration of about 0-10%, 0.5-9.5%, 1-9%, 1.5-8.5%, 2-8%, 3- 8%, 4-8%, 5-8%, 6-8%, 7-8%, 2.5-7.5%, 3-7%, 3.5-6.5%, 4-6%, or 4.5-5.5% v / v. In some embodiments, a pharmaceutical composition includes HSA at a concentration of about 0%, 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10.0% w / v.

[0271] In some embodiments, a pharmaceutical composition includes trehalose at a concentration of about 0-500 mM, 50-450 mM, 100-400 mM, 150-350 mM, or 200-300 mM. In some embodiments, a pharmaceutical composition includes trehalose at a concentration of about 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, or 500 mM.

[0272] Exemplary pharmaceutical composition components are shown in Table 1.Table 1. Exemplary pharmaceutical composition components*HSA in addition to PlasmaLyte.

[0273] In some embodiments, a pharmaceutical composition comprises hypoimmunogenic cells described herein and a pharmaceutically acceptable carrier comprising 31.25% (v / v) Plasma-Lyte A, 31.25% (v / v) of 5% dextrose / 0.45% sodium chloride, 10% dextran 40 (LMD) / 5% dextrose, 20% (v / v) of 25% human serum albumin (HSA), and 7.5% (v / v) dimethyl sulfoxide (DMSO).

[0274] In some embodiments, a pharmaceutical composition comprises hypoimmunogenic cells described herein and a pharmaceutically acceptable carrier comprising 75% Cryostor CS10, 25% (v / v) Plasma-Lyte A, 7.5% (v / v) DMSO, and 1.2% (v / v) HSA.

[0275] In some embodiments, a pharmaceutical composition comprises hypoimmunogenic cells described herein and a pharmaceutically acceptable carrier comprising 31.25% (v / v) Plasma-Lyte A, 31.25% (v / v) of 5% dextrose / 0.45% sodium chloride, 10% dextran 40 (LMD) / 5% dextrose, 20% (w / v) of 25% human serum albumin (HSA), and 7.5% (v / v) dimethyl sulfoxide (DMSO).

[0276] In some embodiments, a pharmaceutical composition comprises hypoimmunogenic cells described herein and a pharmaceutically acceptable carrier comprising 75% Cryostor CS10, 25% (v / v) Plasma-Lyte A, 7.5% (v / v) DMSO, and 1.2% (w / v) HSA.3. Diseases, Disorders and Conditions to be TreatedPost-transplant disorders encompass a group of disorders that can arise in patients after transplant of a solid organ, tissue, cell, or limb. Exemplary post-transplant disorders that can arise as a result of an immunosuppressive therapy in a post-transplant patient include antibody-mediated rejection (AMR) and post-transplant lymphoproliferative disorder (PTLD). AMR results from the emergence of graft-directed antibodies, which often precede subsequent graft rejection and graft failure. In turn, graft failure can have serious implications for patient survival. PTLD results from long-term immunosuppression in patients who have received transplants and the risk for PTLD has been shown to correlatewith the level of immunosuppression. Most PTLD cases are of B cell origin (> 85-90%) and although CAR T cell therapy has shown remarkable efficacy in non-transplant patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL), its use in transplant patients is jeopardized by immunosuppression. In some embodiments, a subject of the present disclosure has received a solid organ transplant. In some embodiments, a solid organ transplant comprises a kidney, pancreas, heart, lung, liver, stomach, uterus and / or intestine transplant. In some embodiments, a subject of the present disclosure has received a tissue transplant. In some embodiments, a tissue transplant comprises a cornea, bone, tendon, skin, heart valve, nerve and / or vein transplant. In some embodiments, a subject of the present disclosure has received a cell transplant. In some embodiments, a cell transplant comprises a bone marrow, stem cell, and / or islet cell transplant. In some embodiments, an islet cell transplant comprises transplantation of alpha, beta, and / or delta islet cells. In some embodiments, a subject of the present disclosure has received a limb transplant. In some embodiments, a limb transplant comprises a hand, arm and / or foot transplant.

[0277] Autoimmune or inflammatory disorders include diseases or disorders arising from and directed against an individual's own tissues or organs or a manifestation thereof or a condition resulting therefrom. In one embodiment, it refers to a condition that results from, or is aggravated by, the production of T cells that are reactive with normal body tissues and antigens. In one embodiment, it refers to a condition that results from, or is aggravated by, the production by antibodies that are reactive with normal body tissues and antigens.

[0278] In some embodiments, an autoimmune disease or disorder comprises systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, anti -neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, multiple sclerosis (relapsing and / or progressive), pemphigus vulgaris, autoimmune blistering skin diseases, membranous nephropathy, anti-NMDA receptor neuropathy, neuromyelitis optica, idiopathic thrombocytopenic purpura, autoimmune hepatitis, type 1 diabetes mellitus, rheumatoid arthritisjuvenile rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy, polymyositis / dermatomyositis, stiff persons disease, anti-NMDA receptor encephalitis, anti-synthetase autoimmune syndromes, antiphospholipid antibody syndrome, Sjogren’s syndrome, cryoglobulinemia, focal segmental glomerulosclerosis, rapidly progressive glomerulopathy, autoimmune hemolytic anemia, amyloidosis, scleroderma, idiopathic inflammatory myositis, or immune-mediated necrotizing myopathy.

[0279] In some embodiments, a subject of the present disclosure has or is suspected of having a post-transplant disorder or an autoimmune disorder. In some embodiments, a post-transplant disorder comprises antibody-mediated rejection (AMR). In some embodiments, a post-transplant disorder comprises post-transplant lymphoproliferative disorder (PTLD).

[0280] In some embodiments, a subject of the present disclosure has a suppressed immune system. In some embodiments, a subject of the present disclosure has been treated with an immunosuppressive and / or immunomodulatory therapy. Non-limiting examples of an immunosuppressive and / or immunomodulatory therapy include cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15- deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti -thymocyte globulin, thymopentin, thymosin-a and similar agents. In some embodiments, the immunosuppressive and / or immunomodulatory agent is selected from a group of immunosuppressive antibodies consisting of antibodies binding to p75 of the IL-2 receptor, antibodies binding to, for instance, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-gamma, TNF -alpha, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL- 10, CDlla, or CD58, and antibodies binding to any of their ligands. In some embodiments, an immunosuppressive therapy comprises an FKBP inhibitor. In some embodiments, an FKBP inhibitor comprises rapamycin. In some embodiments, an FKBP inhibitor comprises a rapalog. In some embodiments, an FKBP inhibitor comprises Tacrolimus. In some embodiments, an immunosuppressive therapy comprises a cyclophilin A inhibitor. In some embodiments, a cyclophilin A inhibitor comprises Cyclosporine A, Voclosporin, or a Cyclosporine analogue.D. Characteristics of Hypoimmunogenic Cells

[0281] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of immune activation in the subject or patient. In some instances, a level of immune activation elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of immune activation produced by the administration of immunogeniccells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit immune activation in the subject or patient.

[0282] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of T cell response in the subject or patient. In some instances, a level of T cell response elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of T cell response produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit a T cell response to the cells in the subject or patient.

[0283] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of NK cell response in the subject or patient. In some instances, a level of NK cell response elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of NK cell response produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit an NK cell response to the cells in the subject or patient.

[0284] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of macrophage engulfment in the subject or patient. In some instances, a level of NK cell response elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of macrophage engulfment produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit macrophage engulfment of the cells in the subject or patient.

[0285] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of systemic TH1 activation in the subject or patient. In some instances, a level of systemic TH1 activation elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of systemic TH1 activation produced by the administrationof immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit systemic TH1 activation in the subject or patient.

[0286] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of NK cell killing in the subject or patient. In some instances, a level of NK cell killing elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of NK cell killing produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit NK cell killing in the subject or patient.

[0287] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of immune activation of peripheral blood mononuclear cells (PBMCs) in the subject or patient. In some instances, a level of immune activation of PBMCs elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of immune activation of PBMCs produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit immune activation of PBMCs in the subject or patient.

[0288] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of donor-specific IgG antibodies in the subject or patient. In some instances, a level of donor-specific IgG antibodies elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of donor-specific IgG antibodies produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit donor-specific IgG antibodies in the subject or patient.

[0289] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of donor-specific IgM antibodies in the subject or patient. In some instances, a level of donor-specific IgM antibodies elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% lower compared to the level of donor-specific IgM antibodies produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit donor-specific IgM antibodies in the subject or patient.

[0290] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of IgM and IgG antibody production in the subject or patient. In some instances, a level of IgM and IgG antibody production elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of IgM and IgG antibody production produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit IgM and IgG antibody production in the subject or patient.

[0291] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of cytotoxic T cell killing in the subject or patient. In some instances, a level of cytotoxic T cell killing elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of cytotoxic T cell killing produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit cytotoxic T cell killing in the subject or patient.

[0292] In some embodiments, an administered population of hypoimmunogenic cells such as hypoimmunogenic CAR-T cells elicits a decreased or lower level of complementdependent cytotoxicity (CDC) in the subject or patient. In some instances, a level of CDC elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the level of CDC produced by the administration of immunogenic cells. In some embodiments, an administered population of hypoimmunogenic cells fails to elicit CDC in the subject or patient.E. Assays for Hypoimmunogenicity Phenotypes

[0293] Once the hypoimmunogenic cells have been generated, they may be assayed for their hypoimmunogenicity as is described in W02016183041 and WO2018132783.

[0294] In some embodiments, hypoimmunogenicity is assayed using a number of techniques as exemplified in Figure 13 and Figure 15 of WO2018132783. These techniques include transplantation into allogeneic hosts and monitoring for cell growth and / or persistence that escape the host immune system. Similarly, the T cell and / or B cell response of the host animal to such cells are tested to confirm that the cells do not cause an immune reaction in the host animal. T cell responses can be assessed by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF). B cell responses or antibody responses are assessed using FACS or Luminex. Additionally or alternatively, the cells may be assayed for their ability to avoid innate immune responses, e.g., NK cell killing, as is generally shown in Figures 14 and 15 of WO2018132783.

[0295]

[0296]

[0297] Additional techniques for determining immunogenicity including hypoimmunogenicity of the cells are described in, for example, Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, the disclosures including the figures, figure legends, and description of methods are incorporated herein by reference in their entirety.

[0298] As will be appreciated by those in the art, the successful reduction of the MHC I function (HLA I when the cells are derived from human cells) in the T cells can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind the HLA complex; for example, using commercially available HLA-A, HLA-B, and HLA-C antibodies that bind to the alpha chain of the human major histocompatibility HLA Class I antigens.

[0299] In addition, the cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This may be assayed by FACS analysis using antibodies to one or more HLA cell surface components as discussed above.

[0300] The successful reduction of the MHC II function (HLA II when the cells are derived from human cells) in the T cells can be measured using techniques known in the art such as Western blotting using antibodies to the protein, FACS techniques, RT-PCR techniques, etc.

[0301] In addition, the cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is done as is known in the art (See Figure 21of WO2018132783, for example) and generally is done using either Western Blots or FACS analysis based on commercial antibodies that bind to human HL A Class II HLA-DR, DP and most DQ antigens.

[0302] In addition to the reduction of HLA I and II (or MHC I and II), the hypoimmunogenic cells of the technology have a reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting hypoimmunogenic cells “escape” the immune macrophage and innate pathways due to reduction or lack of the TCR complex and the expression of one or more CD47 transgenes.F. Gene Editing Systems

[0303] Methods for modulating expression of genes and factors (proteins) include genome editing technologies, RNA or protein expression technologies, and the like. For all of these technologies, well known recombinant techniques are used, to generate recombinant nucleic acids as outlined herein.

[0304] In some embodiments, the cells (e.g., primary T cell or CAR-T cell) possess genetic modifications that inactivate the B2M and CIITA genes and express a plurality of exogenous polypeptides selected from the group including CD47 and DUX4, CD47 and CD24, CD47 and CD27, CD47 and CD46, CD47 and CD55, CD47 and CD59, CD47 and CD200, CD47 and HLA-C, CD47 and HLA-E, CD47 and HLA-E heavy chain, CD47 and HLA-G, CD47 and PD-L1, CD47 and IDO1, CD47 and CTLA4-Ig, CD47 and Cl -Inhibitor, CD47 and IL-10, CD47 and IL-35, CD47 and IL-39, CD47 and FasL, CD47 and CCL21, CD47 and CCL22, CD47 and Mfge8, and CD47 and Serpinb9, and any combination thereof. In some instances, such cells also possess a genetic modification that inactivates the CD142 gene.

[0305] In some instances, a gene editing system such as the CRISPR / Cas system is used to facilitate the insertion of tolerogenic factors, such as the tolerogenic factors into a safe harbor or target locus, such as the AAVS1 locus, to actively inhibit immune rejection. In some instances, the tolerogenic factors are inserted into a safe harbor or target locus using an expression vector. In some embodiments, the safe harbor or target locus is an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91), HMGB1, ABO, RHD, FUT1, or KDM5D gene locus.

[0306] In some embodiments, expression of a target gene (e.g., DUX4, CD47, or another tolerogenic factor gene) is increased by expression of fusion protein or a protein complex containing (1) a site-specific binding domain specific for the endogenous target gene e.g., DUX4, CD47, or another tolerogenic factor gene) and (2) a transcriptional activator.

[0307] In some embodiments, the regulatory factor is comprised of a site specific DNA-binding nucleic acid molecule, such as a guide RNA (gRNA). In some embodiments, the method is achieved by site specific DNA-binding targeted proteins, such as zinc finger proteins (ZFP) or fusion proteins containing ZFP, which are also known as zinc finger nucleases (ZFNs).

[0308] In some embodiments, the regulatory factor comprises a site-specific binding domain, such as using a DNA binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to the gene at a targeted region. In some embodiments, the provided polynucleotides or polypeptides are coupled to or complexed with a site-specific nuclease, such as a modified nuclease. For example, in some embodiments, the administration is effected using a fusion comprising a DNA-targeting protein of a modified nuclease, such as a meganuclease or an RNA-guided nuclease such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, such as CRISPR-Cas9 system. In some embodiments, the nuclease is modified to lack nuclease activity. In some embodiments, the modified nuclease is a catalytically dead dCas9.

[0309] In some embodiments, the site specific binding domain may be derived from a nuclease. For example, the recognition sequences of homing endonucleases and meganucleases such as I-Scel, I-Ceul, PI-PspI, Pl-Sce, 1-SceIV, I-CsmI, I-PanI, I-Scell, I- Ppol, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al. , (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al., (1989) Gene 82:115-118; Perler et al, (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al., (1996) J. Mol. Biol. 263: 163-180; Argast et al, (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, Chevalier et al, (2002) Molec. Cell 10:895- 905; Epinat et al, (2003) Nucleic Acids Res. 31 :2952-2962; Ashworth et al, (2006) Nature 441 :656-659; Paques et al, (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 2007 / 0117128.

[0310] Zinc finger, TALE, and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and / or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496 and U.S. Publication No. 20110301073.

[0311] In some embodiments, the site-specific binding domain comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds DNA in a sequence-specific manner through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.

[0312] Among the ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632- 637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties.

[0313] Many gene-specific engineered zinc fingers are available commercially. For example, Sangamo Biosciences (Richmond, CA, USA) has developed a platform (CompoZr) for zinc-finger construction in partnership with Sigma-Aldrich (St. Louis, MO, USA),allowing investigators to bypass zinc-finger construction and validation altogether, and provides specifically targeted zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or are custom designed.

[0314] In some embodiments, the site-specific binding domain comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein.

[0315] In some embodiments, the site-specific binding domain is derived from the CRISPR / Cas system. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system, or a “targeting sequence”), and / or other sequences and transcripts from a CRISPR locus.

[0316] In general, a guide sequence includes a targeting domain comprising a polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid.

[0317] In some embodiments, the target site is upstream of a transcription initiation site of the target gene. In some embodiments, the target site is adjacent to a transcription initiation site of the gene. In some embodiments, the target site is adjacent to an RNA polymerase pause site downstream of a transcription initiation site of the gene.

[0318] In some embodiments, the targeting domain is configured to target the promoter region of the target gene to promote transcription initiation, binding of one or more transcription enhancers or activators, and / or RNA polymerase. One or more gRNA can be used to target the promoter region of the gene. In some embodiments, one or more regions of the gene can be targeted. In certain aspects, the target sites are within 600 base pairs on either side of a transcription start site (TSS) of the gene.

[0319] It is within the level of a skilled artisan to design or identify a gRNA sequence that is or comprises a sequence targeting a gene, including the exon sequence and sequences of regulatory regions, including promoters and activators. A genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) target sequences in constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com / gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11 :783-4; www.e-crisp.org / E-CRISP / ; crispr.mit.edu / ). In some embodiments, the gRNA sequence is or comprises a sequence with minimal off-target binding to a nontarget gene.

[0320] In some embodiments, the regulatory factor further comprises a functional domain, e.g., a transcriptional activator.

[0321] In some embodiments, the transcriptional activator is or contains one or more regulatory elements, such as one or more transcriptional control elements of a target gene, whereby a site-specific domain as provided above is recognized to drive expression of such gene. In some embodiments, the transcriptional activator drives expression of the target gene. In some cases, the transcriptional activator, can be or contain all or a portion of an heterologous transactivation domain. For example, in some embodiments, the transcriptional activator is selected from Herpes simplex-derived transactivation domain, Dnmt3a methyltransferase domain, p65, VP 16, and VP64.

[0322] In some embodiments, the regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).

[0323] In certain embodiments, the regulatory factor further comprises a transcriptional regulatory domain. Common domains include, e.g., transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associatedfactors and modifiers; chromatin associated proteins and their modifiers (e.g., kinases, acetylases and deacetylases); and DNA modifying enzymes (e.g., methyltransferases such as members of the DNMT family (e.g, DNMT1, DNMT3A, DNMT3B, DNMT3L, etc., topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. See, e.g., U.S. Publication No. 2013 / 0253040, incorporated by reference in its entirety herein.

[0324] Suitable domains for achieving activation include the HSV VP 16 activation domain (see, e.g., Hagmann et al, J. Virol. 71, 5952-5962 (1 97)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Bank, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Then 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95: 14623-33), and degron (Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplary activation domains include, Oct 1, Oct-2 A, Spl, AP-2, and CTF1 (Seipel etal, EMBOJ. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr et al, (2000) Mol. Endocrinol. 14:329-347; Collingwood et al, (1999) J. Mol. Endocrinol 23:255-275; Leo et al, (2000) Gene 245: 1-11; Manteuffel- Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna et al, (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al, (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al, (1999) Curr. Opin. Genet. Dev. 9:499-504. Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, Cl, API, ARF-5, -6,-1, and -8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1 , See, for example, Ogawa et al, (2000) Gene 245:21-29; Okanami et al, (1996) Genes Cells 1 :87-99; Goff et al, (1991) Genes Dev. 5:298- 309; Cho et al, (1999) Plant Mol Biol 40:419-429; Ulmason et al, (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al, (2000) Plant J. 22: 1-8; Gong et al, (1999) Plant Mol. Biol. 41 :33-44; and Hobo et al. , (1999) Proc. Natl. Acad. Sci. USA 96: 15,348- 15,353.

[0325] Exemplary repression domains that can be used to make genetic repressors include, but are not limited to, KRAB A / B, KOX, TGF -beta-inducible early gene (TIEG), v- erbA, SID, MBD2, MBD3, members of the DNMT family (e.g, DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and MeCP2. See, for example, Bird et al, (1999) Cell 99:451-454; Tyler et al, (1999) Cell 99:443-446; Knoepfler et al, (1999) Cell 99:447-450; and Robertson et al, (2000) Nature Genet. 25:338-342. Additional exemplary repression domainsinclude, but are not limited to, R0M2 and AtHD2A. See, for example, Chem et al, (1996) Plant Cell 8:305-321; and Wu et al, (2000) Plant J. 22: 19-27.

[0326] In some instances, the domain is involved in epigenetic regulation of a chromosome. In some embodiments, the domain is a histone acetyltransferase (HAT), e.g., type- A, nuclear localized such as MYST family members MOZ, Ybf2 / Sas3, MOF, and Tip60, GNAT family members Gcn5 or pCAF, the p300 family members CBP, p300 or Rttl09 (Bemdsen and Denu (2008) Curr Opin Struct Biol 18(6): 682-689). In other instances the domain is a histone deacetylase (HD AC) such as the class I (HDAC-1, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7 and 9), HD AC IIB (HDAC 6 and 10)), class IV (HDAC-1 1), class III (also known as sirtuins (SIRTs); SIRT1-7) (see Mottamal et al., (2015) Molecules 20(3):3898-3941). Another domain that is used in some embodiments is a histone phosphorylase or kinase, where examples include MSK1, MSK2, ATR, ATM, DNA-PK, Bubl, VprBP, IKK-a, PKCpi, Dik / Zip, JAK2, PKC5, WSTF and CK2. In some embodiments, a methylation domain is used and may be chosen from groups such as Ezh2, PRMT1 / 6, PRMT5 / 7, PRMT 2 / 6, CARMI, set7 / 9, MLL, ALL-1, Suv 39h, G9a, SETDB1, Ezh2, Set2, Doti, PRMT 1 / 6, PRMT 5 / 7, PR-Set7 and Suv4-20h, Domains involved in sumoylation and biotinylation (Lys9, 13, 4, 18 and 12) may also be used in some embodiments (review see Kousarides (2007) Cell 128:693-705).

[0327] Fusion molecules are constructed by methods of cloning and biochemical conjugation that are well known to those of skill in the art. Fusion molecules comprise a DNA-binding domain and a functional domain e.g., a transcriptional activation or repression domain). Fusion molecules also optionally comprise nuclear localization signals (such as, for example, that from the SV40 medium T-antigen) and epitope tags (such as, for example, FLAG and hemagglutinin). Fusion proteins (and nucleic acids encoding them) are designed such that the translational reading frame is preserved among the components of the fusion.

[0328] Fusions between a polypeptide component of a functional domain (or a functional fragment thereof) on the one hand, and a non-protein DNA-binding domain (e.g., antibiotic, intercalator, minor groove binder, nucleic acid) on the other, are constructed by methods of biochemical conjugation known to those of skill in the art. See, for example, the Pierce Chemical Company (Rockford, IL) Catalogue. Methods and compositions for making fusions between a minor groove binder and a polypeptide have been described. Mapp et al, (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Likewise, CRISPR / Cas TFs and nucleasescomprising a sgRNA nucleic acid component in association with a polypeptide component function domain are also known to those of skill in the art and detailed herein.EXAMPLES

[0329] The following examples are provided so as to describe to the skilled artisan how to make and use methods and compositions described herein, and are not intended to limit the scope of the present disclosure.Example 1: Methods

[0330] This Example provides methods useful for generating and testing of engineered cells provided herein.Mouse models

[0331] Female humanized NSG-SGM3 mice (strain# 013062) were purchased from the Jackson Laboratories . Humanized mice were not thymectomized, received human CD34+ cells at 12 weeks of age, and were included into study groups 6 - 8 weeks after humanization. Animals were randomly assigned to experimental groups. The number of animals per experimental group is presented in each figure. Animal experiments were approved by the Explora BioLabs Institutional Animal Care and Use Committee. Animals received humane care and all experiments followed local guidelines. Mice were housed in 12-hour light-dark cycles with humidity between 30-70% at ambient temperature of 68-79 degrees Fahrenheit. The study and control animals were housed in the same room. The animal facility is a specific pathogen-free facility. Euthanasia was conducted via exsanguination under isoflurane anesthesia followed by cervical dislocation.CAR- T cell production, gene editing, and characterization

[0332] Isolated human CD4+ and CD8+ T cells were thawed and activated with CD3 / CD28 CTS Dynabeads (ThermoFisher) for 24 hours in complete CTS OpTimizer Media containing 100 lU / ml human IL-2. Following activation, CD4+ and CD8+ T cells were combined at a 1 : 1 ratio and plated in 12-well non-TC treated plates at 5 million cells / ml in CTS OpTmizer media containing 100 lU / ml human IL-2. In each well, 5 million total cells were mixed with VSV-G pseudotyped lentiviral vectors packaging CD19CAR or CD47- CD19CAR transgenes at a final MOI of 11 and spinfected at 1000g for 60 minutes at 32 degrees Celsius. Following centrifugation, pl...

Claims

CLAIMS1. An engineered T cell comprising modifications that:(a) reduce expression of FKBP12,(b) reduce expression of(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules,(c) increase expression of a tolerogenic factor, and(d) express a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modifications or a wild-type T cell.

2. The engineered T cell of claim 1, wherein the modification that reduces expression of FKBP12 inactivates or disrupts one or more alleles of FKBP12.

3. The engineered T cell of claim 2, wherein the modification that inactivates or disrupts one or more alleles of FKBP12 comprises: inactivation or disruption of one allele of the FKBP12 gene; inactivation or disruption of both alleles of the FKBP12 gene; or inactivation or disruption of all FKBP12 coding alleles in the engineered T cell.

4. The engineered T cell of claim 2 or 3, wherein the modification that inactivates or disrupts one or more alleles of FKBP12 comprises an indel in the FKBP12 gene.

5. The engineered T cell of any one of claims 2-4, wherein the modification that inactivates or disrupts one or more alleles of FKBP12 comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the FKBP12 gene.

6. An engineered T cell comprising modifications that:(a) reduce expression of cyclophilin A,(b) reduce expression of:(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules,(c) increase expression of a tolerogenic factor, and(d) express a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modifications or a wild-type T cell.

7. The engineered T cell of claim 6, wherein the modification that reduces expression of cyclophilin A inactivates or disrupts one or more alleles of cyclophilin A.

8. The engineered T cell of claim 7, wherein the modification that inactivates or disrupts one or more alleles of cyclophilin A comprises: inactivation or disruption of one allele of the cyclophilin A gene; inactivation or disruption of both alleles of the cyclophilin A gene; or inactivation or disruption of all cyclophilin A coding alleles in the engineered T cell.

9. The engineered T cell of claim 7 or 8, wherein the modification that inactivates or disrupts one or more alleles of cyclophilin A comprises an indel in the cyclophilin A gene.

10. The engineered T cell of any one of claims 6-9, wherein the modification that inactivates or disrupts one or more alleles of cyclophilin A comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the cyclophilin A gene.

11. The engineered T cell of any one of claims 1-10, wherein (b) comprises one or more modifications that inactivate or disrupt one or more alleles of:(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules.

12. The engineered T cell of any one of claims 1-11, wherein the T cell further comprises one or more modifications that reduce expression of T cell receptor alpha constant (TRAC) and / or T cell receptor beta constant (TRBC) relative to a comparable T cell or wild-type T cell.

13. The engineered T cell of any one of claims 1-12, wherein the T cell further comprises one or more modifications that knock-out expression of T cell receptor alpha constant (TRAC) and / or T cell receptor beta constant (TRBC) relative to a comparable T cell or wildtype T cell.

14. The engineered T cell of any one of claims 1-13, wherein the T cell comprises reduced expression of beta-2-microglobulin (B2M) and / or Class II Major Histocompatibility Complex Transactivator (CIITA) relative to a comparable T cell or wild-type T cell.

15. A pharmaceutical composition comprising a population of engineered T cells comprising:(a) reduced expression of FKBP12,(b) reduced expression of:(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules,(c) increased expression of a tolerogenic factor, and(d) expression a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modifications or a wild-type T cell.

16. A pharmaceutical composition comprising a population of engineered T cells comprising:(a) reduced expression of cyclophilin A,(b) reduced expression of:(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules,(c) increased expression of a tolerogenic factor, and(d) expression a chimeric antigen receptor (CAR), wherein the reduced expression of (a) and (b) and increased expression of (c) are relative to a comparable T cell that does not comprise the modifications or a wild-type T cell.

17. A method of making a population of engineered T cells, the method comprising:(a) reducing expression of FKBP12 in a population of T cells relative to comparable T cells or wild-type T cells, and(b) expressing a chimeric antigen receptor (CAR) in the population of T cells, thereby making a population of engineered T cells.

18. A method of making a population of engineered T cells, the method comprising:(a) reducing expression of cyclophilin A in a population of T cells relative to comparable T cells or wild-type T cells, and(b) expressing a chimeric antigen receptor (CAR) in the population of T cells, thereby making a population of engineered T cells.

19. A method comprising administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that:(a) reduce expression of FKBP12 relative to comparable T cells or wild-type T cells, and(b) express a chimeric antigen receptor (CAR).

20. A method comprising administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that:(a) reduce expression of cyclophilin A relative to comparable T cells or wild-type T cells, and(b) express a chimeric antigen receptor (CAR).

21. The method of any one of claims 17-20, wherein the engineered T cells comprise one or more modifications that reduce expression of one or more alleles of:(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules.

22. The method of any one of claims 17-21, wherein the engineered T cells comprise one or more modifications that inactivate or disrupt one or more alleles of:(i) one or more major histocompatibility complex (MHC) class I molecules and / or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(ii) one or more MHC class II molecules and / or one or more molecules that regulate expression of the one or more MHC class II molecules.

23. The method of any one of claims 17-22, wherein the engineered T cells comprise one or more modifications that reduce expression of beta-2-microglobulin (B2M), Class II Major Histocompatibility Complex Transactivator (CIITA), and T cell receptor alpha constant (TRAC) relative to a comparable T cell or wild-type T cell.

24. The method of any one of claims 17-23, wherein the engineered T cells comprise a modification that increases expression of a tolerogenic factor, wherein the increased expression is relative to a comparable T cell or wild-type T cell.

25. The method of any one of claims 19-24, wherein the method is a method of treating or preventing a post-transplant disorder.

26. The method of claim 25, wherein the post-transplant disorder comprises antibody- mediated rejection (AMR).

27. The method of claim 25, wherein the post-transplant disorder comprises a posttransplant lymphoproliferative disorder (PTLD).

28. A method of treating or preventing a post-transplant disorder, the method comprising administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that:(a) reduce expression of FKBP12 relative to comparable T cells or wild-type T cells, or(b) reduce expression of cyclophilin A relative to comparable T cells or wild-type T cells; and(c) express a chimeric antigen receptor (CAR).

29. A method of treating or preventing a post-transplant disorder, the method comprising administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that:(a) reduce expression of FKBP12 relative to comparable T cells or wild-type T cells, or(b) reduce expression of cyclophilin A relative to comparable T cells or wild-type T cells; and(c) express a chimeric antigen receptor (CAR);(d) reduce expression of beta-2-microglobulin (B2M), Class II Major Histocompatibility Complex Transactivator (CIITA), and T cell receptor alpha constant (TRAC) relative to a comparable T cell or wild-type T cell; and(e) increase expression of a tolerogenic factor, wherein the increased expression is relative to a comparable T cell or wild-type T cell.

30. The method of claim 28 or 29, wherein the post-transplant disorder comprises antibody-mediated rejection (AMR).

31. The method of claim 28 or 29, wherein the post-transplant disorder comprises a posttransplant lymphoproliferative disorder (PTLD).

32. A method of treating or preventing an autoimmune disease or disorder, the method comprising administering to a subject a composition comprising a population of engineered T cells, wherein the engineered T cells comprise one or more modifications that:(a) reduce expression of FKBP12 relative to comparable T cells or wild-type T cells, or(b) reduce expression of cyclophilin A relative to comparable T cells or wild-type T cells; and(b) express a chimeric antigen receptor (CAR).

33. The method of claim 32, wherein the autoimmune disease or disorder comprises systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, anti-neutrophilic cytoplasmic autoantibody (ANCA) associated vasculitis, granulomatous polyangiitis, microscopic polyangiitis, multiple sclerosis (relapsing and / or progressive), pemphigus vulgaris, autoimmune blistering skin diseases, membranous nephropathy, anti -NMD A receptor neuropathy, neuromyelitis optica, idiopathic thrombocytopenic purpura, autoimmune hepatitis, type 1 diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy, polymyositis / dermatomyositis, stiff persons disease, anti -NMD A receptor encephalitis, anti-synthetase autoimmune syndromes, antiphospholipid antibody syndrome, Sjogren’s syndrome, cryoglobulinemia, focal segmental glomerulosclerosis, rapidly progressive glomerulopathy, autoimmune hemolytic anemia, amyloidosis, scleroderma, idiopathic inflammatory myositis, or immune-mediated necrotizing myopathy.

34. The method of any one of claims 19-33, wherein the subject has a suppressed immune system.

35. The method of any one of claims 19-34, wherein the subject has received an immunosuppressive therapy.

36. The method of claim 35, wherein the immunosuppressive therapy comprises an FKBP inhibitor.

37. The method of claim 36, wherein the FKBP inhibitor comprises rapamycin or an analogue thereof.

38. The method of claim 37, wherein the analogue is a rapalog.

39. The method of claim 36, wherein the FKBP inhibitor comprises tacrolimus or an analogue thereof.

40. The method of any one of claims 35-39, wherein the immunosuppressive therapy is a combination of rapamycin and tacrolimus.

41. The method of claim 35, wherein the immunosuppressive therapy comprises a cyclophilin A inhibitor.

42. The method of claim 41, wherein the cyclophilin A inhibitor comprises Cyclosporine A, Voclosporin, or an analogue thereof.

43. The method of any one of claims 4-42, wherein the subject has received a solid organ transplant.

44. The method of claim 43, wherein the solid organ transplant comprises a kidney, pancreas, heart, lung, liver, stomach, uterus and / or intestine transplant.

45. The method of any one of claims 19-42, wherein the subject has received a tissue transplant.

46. The method of claim 45, wherein the tissue transplant comprises a cornea, bone, tendon, skin, heart valve, nerve and / or vein transplant.

47. The method of any one of claims 19-42, wherein the subject has received a cell transplant.

48. The method of claim 47, wherein the cell transplant comprises a bone marrow, stem cell, and / or islet cell transplant.

49. The method of claim 48, wherein the islet cell transplant comprises transplantation of alpha, beta, and / or delta islet cells.

50. The method of any one of claims 19-42, wherein the subject has received a limb transplant.

51. The method of claim 50, wherein the limb transplant comprises a hand, arm and / or foot transplant.

52. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the engineered T cells comprise one or more modifications that inactivate or disrupt one or more alleles of one or more T-cell receptor (TCR) molecules and / or one or more molecules that regulate expression of the one or more TCR molecules.

53. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the CAR is or comprises a CDlO-specific CAR, a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD24-specific CAR, a CD27-specific CAR, a CD30-specific CAR, a CD38-specific CAR, a CD45R-specific CAR, a CD138- specific CAR, a CD319-specific CAR, GPRC5D-specific CAR, a CD70-specific CAR, a CD79b-specific CAR, or an EBV antigen-specific CAR, or a BCMA-specific CAR.

54. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the CAR comprises an antigen binding domain that binds to CD 19, CD20, CD22, BCMA, GPRC5D, CD10, CD24, CD27, CD30, CD38, CD45R, CD138, CD319, CD70, CD79b, EBNA1, LMP1, LMP2, LMP2A, EBNA3A, EBNA3C, BZLF1, BMLF1, gp350, or gH / gL.

55. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the CAR comprises an antigen binding domain that binds to a ligand expressed on B cells, plasma cells and / or plasmablasts.

56. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the CAR results in B cell depletion.

57. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the tolerogenic factor comprises CD47, A20 / TNFAIP3, B2M-HLA- E, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD64,CD200, CCL21, CCL22, CTLA4-Ig, Cl inhibitor, CR1, DUX4, FASL, HLA-C, HLA-E, HLA-E heavy chain, HLA-F, HLA-G, H2-M3, ID01, IL-10, IL15-RF, IL-35, IL-39, MANF, Mfge8, PD-L1, Serpinb9, or any combination thereof.

58. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the tolerogenic factor comprises CD47.

59. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the engineered T cell or pharmaceutical composition is delivered as a combination therapy with an immunosuppressive therapy.

60. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the combination therapy comprises simultaneous delivery of the engineered T cell or pharmaceutical composition and the immunosuppressive therapy.

61. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the combination therapy comprises sequential delivery of the engineered T cell or pharmaceutical composition and the immunosuppressive therapy.

62. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce expression of the one or more MHC class I molecules, the one or more MHC class II molecules, and / or the one or more TCR molecules in the hypoimmunogenic T cells, relative to comparable or wild-type T cells.

63. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein expression of the one or more MHC class I molecules, the one or more MHC class II molecules, and the one or more TCR molecules is reduced in the hypoimmunogenic T cells relative to the comparable or wild-type T cell.

64. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the comparable or wild-type T cells do not comprise one or more of the modifications.

65. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce cell surface expression of the one or more MHC class I molecules relative to the comparable or wild-type T cell.

66. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce cell surface trafficking of the one or more MHC class I molecules relative to the comparable or wild-type T cell.

67. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce a function of the one or more MHC class I molecules.

68. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more MHC class I molecules is one or more human leukocyte antigen (HLA) class I molecules.

69. The engineered T cell, pharmaceutical composition, or method of claim 68, wherein the one or more HLA class I molecules is HLA-A, HLA-B, HLA-C, or a combination thereof.

70. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more molecules that regulate cell surface expression of the one or more MHC class I molecules, regulate cell surface trafficking of the one or more MHC class I molecules, and / or reduce a function of the one or more MHC class I molecules are beta-2 microglobulin (B2M).

71. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications comprise a modification that inactivates or disrupts one or more alleles of B2M.

72. The engineered T cell, pharmaceutical composition, or method of claim 71, wherein the modification that inactivates or disrupts one or more alleles of B2M reduces mRNA expression of the B2M gene.

73. The engineered T cell, pharmaceutical composition, or method of claim 71 or 72, wherein the modification that inactivates or disrupts one or more alleles of B2M reduces protein expression of B2M.

74. The engineered T cell, pharmaceutical composition, or method of any one of claims 71-73, wherein the modification that inactivates or disrupts one or more alleles of B2M comprises: inactivation or disruption of one allele of the B2M gene; inactivation or disruption of both alleles of the B2M gene; or inactivation or disruption of all B2M coding alleles in the engineered T cell.

75. The engineered T cell, pharmaceutical composition, or method of any one of claims 71-74, wherein the modification that inactivates or disrupts one or more alleles of B2M comprises an indel in the B2M gene.

76. The engineered T cell, pharmaceutical composition, or method of any one of claims 71-75, wherein the modification that inactivates or disrupts one or more alleles of B2M comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.

77. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce cell surface expression of the one or more MHC class II molecules relative to the comparable or wild-type T cell.

78. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce cell surface trafficking of the one or more MHC class II molecules relative to the comparable or wild-type T cell.

79. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce a function of the one or more MHC class II molecules.

80. The engineered T cell, pharmaceutical composition, or method of claim 79, wherein the function of the one more MHC class II molecules is antigen presentation.

81. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more MHC class II molecules is one or more human leukocyte antigen (HLA) class II molecules.

82. The engineered T cell, pharmaceutical composition, or method of claim 81, wherein the one or more HLA class II molecules is HLA-DP, HLA-DQ, HLA-DR, or a combination thereof.

83. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more molecules that regulate expression of the one or more MHC class II molecules are Class II Major Histocompatibility Complex Transactivator (CIITA).

84. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications comprise a modification that inactivates or disrupts one or more alleles of CIITA.

85. The engineered T cell, pharmaceutical composition, or method of claim 84, wherein the modification that inactivates or disrupts one or more alleles of CIITA reduces mRNA expression of the CIITA gene.

86. The engineered T cell, pharmaceutical composition, or method of claim 84 or 85, wherein the modification that inactivates or disrupts one or more alleles of CIITA reduces protein expression of CIITA.

87. The engineered T cell, pharmaceutical composition, or method of any one of claims 84-86, wherein the modification that inactivates or disrupts one or more alleles of CIITA comprises: inactivation or disruption of one allele of the CIITA gene; inactivation or disruption of both alleles of the CIITA gene; or inactivation or disruption of all CIITA coding alleles in the engineered T cell.

88. The engineered T cell, pharmaceutical composition, or method of any one of claims 84-87, wherein the modification that inactivates or disrupts one or more alleles of CIITA comprises an indel in the CIITA gene.

89. The engineered T cell, pharmaceutical composition, or method of any one of claims 84-88, wherein the modification that inactivates or disrupts one or more alleles of CIITA comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CIITA gene.

90. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the engineered T cell.

91. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce cell surface protein expression of the one or more TCR molecules relative to the comparable or wild-type T cell.

92. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce cell surface trafficking of the one or more TCR molecules relative to the comparable or wild-type T cell.

93. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications reduce a function of the one or more TCR molecules.

94. The engineered T cell, pharmaceutical composition, or method of any claim 93, wherein the function of the one or more TCR molecules is antigen recognition.

95. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more TCR molecules is T cell receptor alpha constant (TRAC), T cell receptor beta constant (TRBC), or a combination thereof.

96. The engineered T cell, pharmaceutical composition, or method of claim 95, wherein the one or more TCR molecules comprise TRAC.

97. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the one or more modifications comprise a modification that inactivates or disrupts one or more alleles of TRAC.

98. The engineered T cell, pharmaceutical composition, or method of claim 97, wherein the modification that inactivates or disrupts one or more alleles of TRAC reduces mRNA expression of the TRAC gene.

99. The engineered T cell, pharmaceutical composition, or method of claim 97 or 98, wherein the modification that inactivates or disrupts one or more alleles of TRAC reduces protein expression of TRAC.

100. The engineered T cell, pharmaceutical composition, or method of any one of claims 97-99, wherein the modification that inactivates or disrupts one or more alleles of TRAC comprises: inactivation or disruption of one allele of the TRAC gene; inactivation or disruption of both alleles of the TRAC gene; or inactivation or disruption of all TRAC coding alleles in the engineered T cell.

101. The engineered T cell, pharmaceutical composition, or method of any one of claims 97-100, wherein the modification inactivation or disruption that inactivates or disrupts one or more alleles of TRAC comprises an indel in the TRAC gene.

102. The engineered T cell, pharmaceutical composition, or method of any one of claims 97-101, wherein the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the TRAC gene.

103. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the tolerogenic factor is encoded by a first exogenous polynucleotide.

104. The engineered T cell, pharmaceutical composition, or method of any one of the previous claims, wherein the CAR is encoded by a second exogenous polynucleotide.