Chimeric antigen receptor (CAR) T cells and related methods for treating autoimmune diseases

Abstract

Disclosed herein are engineered and / or hypoimmunogenic cells, such as engineered stem cells and / or hypoimmunogenic stem cells, engineered cells and / or hypoimmunogenic cells differentiated from such stem cells, engineered CAR-T cells and / or hypoimmunogenic CAR-T cells (differentiated from primary or engineered stem cells and / or hypoimmunogenic stem cells) and related methods of their use and production for use in the treatment of autoimmune and / or inflammatory diseases / disorders. Provided herein are engineered and / or hypoimmunogenic cells that exhibit reduced expression of MHC class I and / or MHC class II human leukocyte antigens and T cell receptors for use in the treatment of autoimmune and / or inflammatory diseases / disorders. In some embodiments, such cells also exogenously express one or more tolerogenic factors, such as CD47, and one or more chimeric antigen receptors (CARS).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 293,637, filed December 23, 2021, and U.S. Provisional Patent Application No. 63 / 320,672, filed March 16, 2022, the contents of which are incorporated herein by reference in their entireties. Summary of the Invention

[0002] Off-the-shelf CAR-T cells and other therapeutic cells may offer advantages over autologous cell-based strategies, including ease of manufacturing, quality control, and avoidance of harmful contamination and T cell dysfunction. However, host-versus-graft immune responses to histo-incompatible T cells are robust and prevent the expansion and persistence of allogeneic CAR-T cells, reducing the efficacy of this approach.

[0003] There is strong evidence in both animal models and human patients that hypoimmunogenic cell transplantation is a scientifically feasible and clinically promising approach for the treatment of many disorders, conditions, and diseases, particularly autoimmune and / or inflammatory diseases / disorders.

[0004] There remains a need for novel approaches, compositions and methods for producing cell-based therapies that avoid detection by the recipient's immune system.

[0005] In some embodiments, provided herein are engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to a wild-type or control cell, the engineered cell further comprising a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of the cell.

[0006] In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In many embodiments, a first exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, a second exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus.

[0007] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at different loci. In many embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the same locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the B2M locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the CIITA locus. In many embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the TRAC locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the TRB locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C (also known as AAVS1) locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus (e.g., the ROSA26 locus), the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. In various embodiments, the safe harbor or target locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26 locus.

[0008] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR, a CD22-specific CAR, and a CD20-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the CAR is a CD22-specific CAR. In some embodiments, the CAR is a CD20-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the CAR is a CD19 / CD20 bispecific CAR. In some embodiments, the CAR is a CD19 / CD22 bispecific CAR.

[0009] In many embodiments, the engineered cells do not express HLA-A, HLA-B, and / or HLA-C antigens. In some embodiments, the engineered cells do not express B2M. In other embodiments, the engineered cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens. In some embodiments, the engineered cells do not express CIITA. In some embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells do not express TCR-alpha and / or TCR-beta.

[0010] In many embodiments, the engineered cells are pluripotent stem cells. In some embodiments, the engineered cells are induced pluripotent stem cells.

[0011] In some embodiments, the engineered cells are differentiated cells derived from induced pluripotent stem cells. In various embodiments, the differentiated cells are selected from the group consisting of NK cells and T cells.

[0012] In some embodiments, the engineered cells are derived from primary T cells. In many embodiments, the cells derived from primary T cells are derived from a pool of T cells that includes primary T cells from one or more donor subjects that are different from the recipient subject.

[0013] In some embodiments, the engineered cells are derived from primary NK cells. In many embodiments, the cells derived from primary NK cells are derived from a pool of NK cells that includes primary NK cells from one or more donor subjects that are different from the recipient subject.

[0014] In some embodiments, the engineered cells retain pluripotency and / or retain the ability to differentiate.

[0015] In many embodiments, after transfer into a first subject, the engineered cells exhibit one or more responses selected from the group consisting of reduced (a) T cell response, (b) NK cell response, and (c) macrophage response, compared to wild-type cells after transfer into a second subject. In some cases, the first subject and the second subject are different subjects. In some cases, the macrophage response is phagocytosis. In various embodiments, after transfer into a subject, the engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject, (b) reduced NK cell killing in the subject, and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into the subject. In many embodiments, after transfer into a subject, the engineered cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject, (b) reduced IgM or IgG antibodies in the subject, and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into the subject.

[0016] In some embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデルIn many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデルIn many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル It is a cell.

[0017] In some embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデルIn many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデルIn many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル It is a cell.

[0018] In some embodiments, the engineered cells comprise a B2M comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル, C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells that contain a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells that contain a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデルIn many embodiments, the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル It is a cell.

[0019] In some embodiments, engineered cells are provided that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type or control cells.

[0020] In some embodiments, the engineered cells do not express HLA-A, HLA-B and / or HLA-C antigens. In many embodiments, the engineered cells do not express CIITA.

[0021] In many embodiments, the engineered cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens, hi some embodiments, the engineered cells do not express B2M.

[0022] In many embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells do not express TCR-alpha. In many embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells do not express TCR-beta.

[0023] In various embodiments, the engineered cells overexpress CD47 relative to wild-type or control cells.

[0024] In some embodiments, the engineered cells are pluripotent stem cells. In many embodiments, the engineered cells are induced pluripotent stem cells.

[0025] In many embodiments, the engineered cells are differentiated cells derived from induced pluripotent stem cells, hi some embodiments, the differentiated cells are selected from the group consisting of NK cells and T cells.

[0026] In many embodiments, the engineered cells are cells derived from primary T cells. In some embodiments, the cells derived from primary T cells are derived from a pool of T cells that includes primary T cells from one or more donor subjects that are different from the recipient subject.

[0027] In various embodiments, the engineered cells retain pluripotency and / or retain differentiation potential.

[0028] In some embodiments, after transfer into a subject, the engineered cells elicit one or more responses selected from the group consisting of: (a) a T cell response, (b) a NK cell response, and (c) a macrophage response that is reduced compared to wild-type cells after transfer into a second subject. In some cases, the first subject and the second subject are different subjects. In some cases, the macrophage response is phagocytosis.

[0029] In various embodiments, after transfer into a subject, the engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject, (b) reduced NK cell killing in the subject, and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into a subject. In many embodiments, after transfer into a subject, the engineered cells induce one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject, (b) reduced IgM or IgG antibodies in the subject, and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into a subject.

[0030] In some embodiments, the engineered cells are B2Mインデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some cases, engineered cells are B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル Primary T cells. In some cases, the engineered cells are B2M cells differentiated from hypoimmunogenic induced pluripotent stem cells. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some cases, the engineered cells are selected from the group consisting of 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, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the engineered cells are selected from the group consisting of 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, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In some cases, the engineered cells are selected from the group consisting of 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, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル Primary T cells. In some cases, the engineered cells are B2M cells differentiated from low-immunogenic pluripotent stem cells. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRACインデル / インデル In some embodiments, the engineered cells are B2M T cells. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In some cases, engineered cells are B2M インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル Primary T cells. In some cases, the engineered cells are B2M cells differentiated from hypoimmunogenic induced pluripotent stem cells. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル T cells.

[0031] In some embodiments, the engineered cells are hypoimmunogenic cells.

[0032] In some embodiments, a pharmaceutical composition is provided comprising any population of engineered cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or excipient.

[0033] In some embodiments, the pharmaceutically acceptable additive, carrier, diluent, or excipient comprises one or more selected from the group consisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, human serum albumin (HSA), dimethyl sulfoxide (DMSO), and combinations thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer. In some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline.

[0034] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, a base solution of CryoStor® CSB at a concentration of about 70-80% w / w, and one or more of about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0035] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, a base solution of PlasmaLyte-A™ at a concentration of about 20-30% w / w, and one or more of about 70-80% w / w CryoStor® CSB, about 0.3-5.3% w / v human serum albumin (HSA), about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0036] In some embodiments, a pharmaceutical composition is provided comprising any population of engineered cells described herein, about 0.3-5.3% w / v human serum albumin (HSA), and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0037] In some embodiments, a pharmaceutical composition is provided comprising any population of engineered cells described herein, about 0-20% v / v dimethyl sulfoxide (DMSO), and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), and about 100-400 mM trehalose.

[0038] In some embodiments, a pharmaceutical composition is provided comprising any population of engineered cells described herein, about 100-400 mM trehalose, and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), and about 0-20% v / v dimethyl sulfoxide (DMSO).

[0039] In some embodiments, the pharmaceutical composition comprises about 75% w / w CryoStor® CSB. In some embodiments, the pharmaceutical composition comprises about 25% w / w PlasmaLyte-A™. In some embodiments, the pharmaceutical composition comprises about 0.3% w / v HSA. In some embodiments, the pharmaceutical composition comprises about 7.5% v / v DMSO.

[0040] In some embodiments, a pharmaceutical composition is provided comprising any population of engineered cells described herein, a base solution of CryoStor® CSB at a concentration of about 75% w / w, about 25% w / w PlasmaLyte-A™, about 0.3% w / v human serum albumin (HSA), and about 7.5% v / v dimethyl sulfoxide (DMSO).

[0041] In some embodiments, the population of engineered cells is at most about 8.0 x 10 8 In many embodiments, the population of engineered cells is at most about 6.0 x 10 cells. 8 In other embodiments, the population of engineered cells is about 1.0 x 10 cells. 6 ~about 2.5x10 8 In some embodiments, the population of engineered cells is about 2.0 x 10 cells. 6 ~Approx. 2.0×10 8 Each cell is an individual cell.

[0042] In various embodiments, the population of engineered cells ranges from about 5 ml to about 80 ml. In many embodiments, the population of engineered cells ranges from about 10 ml to about 70 ml. In some embodiments, the population of engineered cells ranges from about 10 ml to about 50 ml.

[0043] In some embodiments, the composition is formulated for administration in a single dose. In many embodiments, the composition is formulated for administration in up to three doses.

[0044] In some embodiments, the compositions are formulated so that administration of a single dose to a subject requires a duration of about 60 minutes or less. In many embodiments, the compositions are formulated so that administration of a single dose to a subject requires a duration of about 30 minutes or less.

[0045] In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 40% survival in the subject 10 days after administration. In various embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 80% survival in the subject about 2 weeks after administration. In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 100% survival in the subject about 3 weeks after administration. In many embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 150% survival in the subject about 4 weeks after administration.

[0046] In another embodiment, there is provided a dosing regimen for treating a disease or disorder in a subject, the dosing regimen comprising administering a pharmaceutical composition comprising any population of engineered cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1 to 3 doses.

[0047] In some embodiments, the pharmaceutical composition administered is up to about 6.0 x 10 in about 1 to 3 doses. 8 In some embodiments, the pharmaceutical composition administered is about 0.6 x 10 cells in about 1-3 doses. 6 ~Approx. 6.0×10 8 In some embodiments, the pharmaceutical composition administered is about 0.2 x 10 cells per kg of subject body weight in about 1 to 3 doses when the subject weighs 50 kg or less. 6 ~Approx. 5.0×10 6 In some embodiments, the pharmaceutical composition administered is about 0.1 x 10 cells in about 1 to 3 doses if the subject weighs more than 50 kg. 8 ~Approx. 2.5×10 8In some embodiments, the pharmaceutical composition administered is about 2.0 x 10 cells per kg of subject body weight in about 1-3 doses. 6 cells up to approximately 2 × 10 8 Each cell is an individual cell.

[0048] In some embodiments, administration of a single dose to a subject takes about 60 minutes or less in duration. In some embodiments, administration of a single dose to a subject takes about 30 minutes or less in duration.

[0049] In some embodiments, the pharmaceutically acceptable additive, carrier, diluent, or excipient is Plasma-Lyte A® ) , dextrose, dextran, sodium chloride, human serum albumin (HSA), dimethyl sulfoxide (DMSO), and combinations thereof.

[0050] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, hi some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline.

[0051] In some embodiments, the population of cells or their progeny are present in the subject for up to 9 months after administration of the pharmaceutical composition, hi some embodiments, the population of cells or their progeny are present in the subject for at least 2 years or more after administration of the pharmaceutical composition.

[0052] In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 40% survival in the subject about 10 days after administration. In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 80% survival in the subject about 2 weeks after administration. In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 100% survival in the subject about 3 weeks after administration. In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 150% survival in the subject about 4 weeks after administration.

[0053] In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 24 hours apart. In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 28 days apart. In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 6 weeks apart. In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 12 months or more apart.

[0054] Provided herein are dosing regimens for treating a disease or disorder in a subject, comprising administering (i) a pharmaceutical composition comprising engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, the engineered cells further comprising a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR). In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the set of exogenous polynucleotides. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis virus glycoprotein (VSV-G) envelope carrying the set of exogenous polynucleotides. In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using a lentiviral-based viral vector. In some embodiments, the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of the cell, and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, the pharmaceutical composition comprises up to about 6.0 x 10 8 Contains cells.

[0055] Provided herein are dosing regimens for treating a disease or disorder in a subject, comprising administering (i) a pharmaceutical composition comprising engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, the engineered cells further comprising a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR). In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using viral transduction. In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using a lentiviral-based viral vector. In some embodiments, the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of the cell; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient. The pharmaceutical composition is administered in 1 to 3 doses.

[0056] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, the engineered cells further comprising a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR), wherein the set of exogenous polynucleotides is inserted into at least one allele of a safe harbor or target locus of the cell; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein a dose of the pharmaceutical composition is administered for a duration of about 60 minutes or less.

[0057] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in a dose range of up to about 6.0 x 10 8 A dosing regimen comprising:

[0058] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in one to three doses.

[0059] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein a dose of the pharmaceutical composition is administered for a duration of about 60 minutes or less.

[0060] In some embodiments, methods of treating an autoimmune disease / disorder and / or an inflammatory disease / disorder in a subject are provided, comprising administering to the subject any of the engineered cells described herein, any of the pharmaceutical compositions described herein, or any of the dosing regimens described herein. In some embodiments, the autoimmune disease / disorder and / or inflammatory disease / disorder is, at least in part, a B cell- and / or plasma cell-mediated autoimmune disease / disorder and / or inflammatory disease / disorder. In some embodiments, the autoimmune disease / disorder and / or inflammatory disease / disorder is a B cell- and / or plasma cell-mediated autoimmune disease / disorder and / or inflammatory disease / disorder. In some embodiments, the B cells and / or plasma cells express CD19, CD20, or a combination thereof. In some embodiments, the autoimmune disease includes, but is not limited to, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes. Further examples of "autoimmune diseases" or "autoimmune disorders" or "inflammatory diseases" or "inflammatory disorders" can be found in Section Z below.

[0061] In some embodiments, methods are provided for preventing T cell exhaustion in a subject, comprising administering to the subject any of the engineered cells described herein, wherein the CAR is a CD19 / CD20 bispecific CAR. In some embodiments, methods are provided for preventing T cell exhaustion in a subject, comprising administering to the subject any of the engineered cells described herein, wherein the CAR is a CD19 / CD22 bispecific CAR.

[0062] In some embodiments, provided herein are methods of preventing T cell exhaustion or treating a disease in a subject, comprising: (i) administering to the subject at a first time a first dosage regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosage regimen comprising a second population of any of the engineered cells described herein, wherein the first dosage regimen and the second dosage regimen are different.

[0063] In some embodiments, provided herein are methods of preventing T cell exhaustion or treating a disease in a subject, the methods comprising: (i) administering to the subject at a first time a first dosing regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosing regimen comprising a second population of any of the engineered cells described herein, wherein both the first population of engineered cells and the second population of engineered cells comprise the same chimeric antigen receptor.

[0064] In some embodiments, provided herein are methods of preventing T cell exhaustion or treating a disease in a subject, comprising: (i) administering to the subject at a first time a first dosing regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosing regimen comprising a second population of any of the engineered cells described herein, wherein the first population of engineered cells and the second population of engineered cells each comprise a different chimeric antigen receptor.

[0065] In some embodiments, provided herein are methods of preventing T cell exhaustion or treating a disease in a subject, the method comprising: (i) administering to the subject at a first time a first dosing regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosing regimen comprising a second population of any of the engineered cells described herein, wherein the engineered cells of the first population comprise a first chimeric antigen receptor that binds a first antigen, and the engineered cells of the second population comprise a second chimeric antigen receptor that binds a second antigen, and wherein the first antigen and the second antigen are the same.

[0066] In some embodiments, provided herein are methods of preventing T cell exhaustion or treating a disease in a subject, comprising: (i) administering to the subject at a first time a first dosing regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosing regimen comprising a second population of any of the engineered cells described herein, wherein the engineered cells of the first population comprise a first chimeric antigen receptor that binds a first antigen, and the engineered cells of the second population comprise a second chimeric antigen receptor that binds a second antigen, and wherein the first antigen and the second antigen are different.

[0067] Provided herein are resting T cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta relative to wild-type T cells, and a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR).

[0068] In some embodiments, the resting T cells are primary T cells. In other embodiments, the resting T cells are differentiated from engineered cells of the present technology.

[0069] In some embodiments, the T cells are CD8 + T cells.

[0070] In some embodiments, the resting T cells have not been treated with an anti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule.

[0071] In some embodiments, the anti-CD3 antibody is OKT3. In some embodiments, the anti-CD28 antibody is CD28.2. In some embodiments, the T cell activating cytokine is selected from the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the soluble T cell costimulatory molecule is selected from the group of soluble T cell costimulatory molecules consisting of anti-CD28 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-CD137L antibody, and anti-ICOS-L antibody.

[0072] In some embodiments, resting T cells do not express activation markers.

[0073] In some embodiments, the inactivated T cells express CD3 and CD28, and CD3 and / or CD28 are inactive.

[0074] In some embodiments, the first exogenous polynucleotide is carried by a lentiviral vector comprising a CD8 binding agent.

[0075] In some embodiments, the resting T cells further comprise a second exogenous polynucleotide encoding CD47.

[0076] In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of a T cell. In some embodiments, the first and / or second exogenous polynucleotide is inserted into at least one allele of a T cell using viral transduction. In some embodiments, the first and / or second exogenous polynucleotide is inserted into at least one allele of a T cell using a lentivirus-based viral vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the first and / or second exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis virus glycoprotein (VSV-G) envelope carrying the first and / or second exogenous polynucleotide. In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 is inserted at a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the first exogenous polynucleotide encoding a CAR is inserted at a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at different loci. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the same locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the B2M locus.In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the CIITA locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the TRAC locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C (also known as AAVS1) locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus (e.g., the ROSA26 locus), the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26 locus.

[0077] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR, a CD22-specific CAR, and a CD20-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD20 bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR.

[0078] In some embodiments, the resting T cells do not express HLA-A, HLA-B, and / or HLA-C antigens. In some embodiments, the resting T cells do not express B2M. In some embodiments, the resting T cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens. In some embodiments, the resting T cells do not express CIITA. In some embodiments, the resting T cells do not express TCR-alpha and TCR-beta.

[0079] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0080] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0081] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0082] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0083] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0084] In some embodiments, the resting T cells comprise a B2M gene encoding a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0085] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0086] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0087] Provided herein are engineered T cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta relative to wild-type T cells, the engineered T cells further comprising a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) delivered by a lentiviral vector. Provided herein are engineered T cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta relative to wild-type T cells, the engineered T cells further comprising a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) delivered by a lentiviral vector comprising a CD8 binding agent.

[0088] In some embodiments, the engineered T cells are primary T cells. In other embodiments, the engineered T cells are differentiated from engineered cells of the present technology. In some embodiments, the T cells are CD8 + T cells.

[0089] In some embodiments, the engineered T cells have not been treated with an anti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule. In some embodiments, the anti-CD3 antibody is OKT3, the anti-CD28 antibody is CD28.2, the T cell activating cytokine is selected from the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21, and the soluble T cell costimulatory molecule is selected from the group of soluble T cell costimulatory molecules consisting of anti-CD28 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-CD137L antibody, and anti-ICOS-L antibody.

[0090] In some embodiments, the engineered T cells do not express activation markers, hi some embodiments, the engineered T cells express CD3 and CD28, and CD3 and / or CD28 are inactive.

[0091] In some embodiments, the engineered T cell further comprises a second exogenous polynucleotide encoding CD47. In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the T cell. In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the first exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into different loci. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the same locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the B2M locus, the CIITA locus, the TRAC locus, the TRB locus, or a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C (also known as AAVS1) locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus (e.g., the ROSA26 locus), the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus.In some embodiments, the safe harbor or target locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26 locus.

[0092] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR, a CD22-specific CAR, and a CD20-specific CAR.

[0093] In some embodiments, the engineered T cells do not express HLA-A, HLA-B, and / or HLA-C antigens, the engineered T cells do not express B2M, the engineered T cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens, the engineered T cells do not express CIITA, and / or the engineered T cells do not express TCR-alpha and TCR-beta.

[0094] In some embodiments, the engineered T cells comprise a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus, the TRB locus, the B2M locus, or the CIITA locus, インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0095] In some embodiments, the rested T cells and / or engineered T cells of the present technology are in a subject, hi other embodiments, the rested T cells and / or engineered T cells of the present technology are in vitro.

[0096] In some embodiments, the resting T cells and / or engineered T cells of the present technology express a CD8 binding agent. In some embodiments, the CD8 binding agent is an anti-CD8 antibody. In some embodiments, the anti-CD8 antibody is selected from the group consisting of a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a human anti-CD8 antibody, a humanized anti-CD8 antibody, a camelid (e.g., llama, alpaca, camel) anti-CD8 antibody, and fragments thereof. In some embodiments, the fragment is an scFV or VHH. In some embodiments, the CD8 binding agent binds to the CD8 alpha chain and / or the CD8 beta chain.

[0097] In some embodiments, the CD8 binding agent is fused to a transmembrane domain incorporated into the viral envelope. In some embodiments, the lentiviral vector is pseudotyped with a viral fusion protein. In some embodiments, the viral fusion protein comprises one or more modifications that reduce binding to its native receptor.

[0098] In some embodiments, the viral fusion protein is fused to a CD8 binding factor. In some embodiments, the viral fusion protein comprises a Nipah virus F glycoprotein and a Nipah virus G glycoprotein fused to a CD8 binding factor. In some embodiments, the lentiviral vector does not comprise a T cell activation molecule or a T cell costimulatory molecule. In some embodiments, the lentiviral vector encodes a first exogenous polynucleotide and / or a second exogenous polynucleotide.

[0099] In some embodiments, after transfer into a first subject, the resting T cells or engineered T cells exhibit one or more responses selected from the group consisting of reduced (a) T cell response, (b) NK cell response, and (c) macrophage response compared to wild-type cells after transfer into a second subject. In some embodiments, the first subject and the second subject are different subjects. In some embodiments, the macrophage response is phagocytosis.

[0100] In some embodiments, after transfer into a subject, the rested T cells or engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject; (b) reduced NK cell killing in the subject; and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into a subject.

[0101] In some embodiments, after transfer into a subject, the rested T cells or engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject; (b) reduced IgM or IgG antibodies in the subject; and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into a subject.

[0102] In some embodiments, resting T cells or engineered T cells are transduced in a subject with a lentiviral vector comprising a CD8 binding agent. In some embodiments, the lentiviral vector carries a gene encoding a CAR and / or CD47.

[0103] Provided herein are pharmaceutical compositions comprising a population of rested T cells and / or engineered T cells of the present technology and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

[0104] Provided herein are methods comprising administering to a subject a composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology.

[0105] In some embodiments, the subject is not administered a T cell activation treatment before, after, and / or concomitantly with administration of the composition, hi some embodiments, the T cell activation treatment comprises lymphodepletion.

[0106] Provided herein are methods of treating a subject suffering from an autoimmune disease / disorder and / or an inflammatory disease / disorder, comprising administering to the subject a composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology, wherein the subject does not receive a T cell activation treatment before, after, and / or concurrently with administration of the composition. In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0107] Provided herein are methods for internally expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising administering to the subject a composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology, wherein the subject is not administered a T cell activation treatment before, after, and / or simultaneously with administration of the composition. In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0108] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology and a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1 to 3 doses.

[0109] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0110] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117 and a CD22 CAR having a CDR sequence of SEQ ID NO: 45, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0111] In some embodiments, the encoded CD19 CAR has the corresponding amino acid sequence set forth in SEQ ID NO: 117 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 SEQ ID NO: 117 and comprises the following components: CD8α signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8α hinge domain, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

[0112] In some embodiments, the encoded CD22 CAR has the corresponding amino acid sequence set forth in SEQ ID NO:45 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 SEQ ID NO:45.

[0113] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0114] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0115] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0116] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0117] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0118] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0119] In some embodiments, provided herein are methods of treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, comprising evaluating the patient for and / or diagnosing the patient with an autoimmune disease and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0120] In some embodiments, the CAR has an scFv sequence of any one of SEQ ID NOs: 19, 29, and 37.

[0121] In some embodiments, the CAR has the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117.

[0122] In some embodiments, the engineered T cells comprise a CD19-specific CAR and a CD20-specific CAR.

[0123] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

[0124] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

[0125] In some embodiments, the CD19 CAR T cells and the CD20 CAR T cells are administered simultaneously.

[0126] In some embodiments, the CD19 CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

[0127] In some embodiments, the CD19 CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

[0128] In some embodiments, the CD20 CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

[0129] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0130] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0131] In some embodiments, the engineered T cells are expanded from primary T cells or their progeny, or are derived from T cells differentiated from iPSCs or their progeny.

[0132] In some embodiments, the engineered T cells are differentiated cells derived from induced pluripotent stem cells or their progeny.

[0133] In some embodiments, the differentiated cells are T cells or natural killer (NK) cells.

[0134] In some embodiments, the engineered T cells are the progeny of a primary immune cell, optionally, the progeny of the primary immune cell is a T cell or an NK cell.

[0135] In some embodiments, the engineered T cells comprise reduced expression of beta-2-microglobulin (B2M) and / or MHC class II transactivator of transcription (CIITA) compared to intact or unmodified wild-type or control cells.

[0136] In some embodiments, the engineered T cells do not express B2M and / or CIITA.

[0137] In some embodiments, the engineered T cells comprise reduced expression of TCR-alpha and / or TCR-beta.

[0138] In some embodiments, the engineered T cells do not express TCR-alpha and / or TCR-beta.

[0139] In some embodiments, the one or more tolerogenic factors are selected from the group consisting of CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, MANF, and Serpinb9.

[0140] In some embodiments, the one or more tolerogenic factors comprises CD47.

[0141] In some embodiments, the CD19-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0142] In some embodiments, the CD19-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0143] In some embodiments, the CD20-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0144] In some embodiments, the CD20-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0145] In some embodiments, one or more of the first, second, and / or third exogenous polynucleotides or the bicistronic polynucleotide are inserted into the first, second, and / or third specific locus of at least one allele of the cell.

[0146] In some embodiments, the first, second, and / or third specific loci are selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0147] In some embodiments, the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus.

[0148] In some embodiments, the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

[0149] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0150] In some embodiments, the gene therapy vector is a retrovirus or a fusosome.

[0151] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using CRISPR / Cas gene editing.

[0152] In some embodiments, the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0153] In some embodiments, the CRISPR / Cas system is selected from the group consisting of: optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; optionally selected from the group consisting of Cas9, Csn2, and Cas4; optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and and Csx10, optionally Csf1, and optionally a Cas effector protein selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d), and optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0154] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0155] In some embodiments, the first, second and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cells using a lentiviral vector.

[0156] In some embodiments, the engineered T cells avoid NK cell-mediated cytotoxicity upon administration to a recipient patient.

[0157] In some embodiments, the engineered T cells are protected from cytolysis by mature NK cells upon administration to a recipient patient.

[0158] In some embodiments, the engineered T cells evade macrophage-mediated cytotoxicity, optionally including phagocytosis and / or reactive oxygen species.

[0159] In some embodiments, the engineered T cells do not induce an immune response against the cells upon administration to a recipient patient.

[0160] In some embodiments, the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, and pulmonary conditions.

[0161] In some embodiments, the administration is selected from the group consisting of intravenous injection, intramuscular injection, intravascular injection, and implantation.

[0162] In some embodiments, the engineered T cells are administered to the patient before, during, or after the initiation of a different therapeutic regimen.

[0163] In some embodiments, the different treatment regimen is selected from the group consisting of re-administration of the same or different cells, and pre-treatment, co-treatment, or post-treatment with an additional agent.

[0164] In some embodiments, the different cells are autologous T cells or NK cells, or CAR-T cells expressing a first CAR that is different from a second CAR expressed by the engineered CAR-T cell.

[0165] In some embodiments, the patient has been treated with an immunodepleting therapy prior to administering the engineered T cells.

[0166] In some embodiments, the immunodepletion therapy comprises administration of fludarabine and / or cyclophosphamide.

[0167] In some embodiments, the patient has previously received antibody therapy.

[0168] In some embodiments, the antibody therapy is rituximab.

[0169] In some embodiments, the immunodepletion therapy is about 1-50 mg / m2 This involves an IV infusion of fludarabine for approximately 1 to 7 days.

[0170] In some embodiments, the immunodepletion therapy is administered at a dose of about 1, about 5, about 10, about 20, about 30, about 40, or about 50 mg / m 2 of fludarabine administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0171] In some embodiments, the immunodepletion therapy is about 30 mg / m 2 This involves an IV infusion of fludarabine for approximately four days.

[0172] In some embodiments, the immunodepletion therapy is about 100-1000 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately 1 to 7 days.

[0173] In some embodiments, the immunodepletion therapy is administered at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 mg / m 2 of cyclophosphamide administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0174] In some embodiments, the immunodepletion therapy is about 500 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately two days.

[0175] In some embodiments, at least about 40×10 4 The engineered T cells are administered to the patient.

[0176] In some embodiments, at least about 40×10 5 The engineered T cells are administered to the patient.

[0177] In some embodiments, the engineered T cells persist in the subject for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0178] In some embodiments, the therapeutic effect of the engineered T cells lasts for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0179] In some embodiments, wild-type or control cells are the starting material.

[0180] In some embodiments, provided herein is a method of using a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequence of SEQ ID NO: 117, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0181] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequence of SEQ ID NO: 117 and a CD22 CAR having the CDR sequence of SEQ ID NO: 45, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0182] In some embodiments, the encoded CD19 CAR has the corresponding amino acid sequence set forth in SEQ ID NO: 117 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 SEQ ID NO: 117 and comprises the following components: CD8α signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8α hinge domain, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

[0183] In some embodiments, the encoded CD22 CAR has the corresponding amino acid sequence set forth in SEQ ID NO:45 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 SEQ ID NO:45.

[0184] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with the disease, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0185] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0186] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0187] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0188] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with the disease, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0189] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0190] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a recipient patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0191] In some embodiments, the CAR has an scFv sequence of any one of SEQ ID NOs: 19, 29, and 37.

[0192] In some embodiments, the CAR has the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117.

[0193] In some embodiments, the engineered T cells comprise a CD19-specific CAR and a CD20-specific CAR.

[0194] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

[0195] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

[0196] In some embodiments, the CD19 CAR T cells and the CD20 CAR T cells are administered simultaneously.

[0197] In some embodiments, the CD19 CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

[0198] In some embodiments, the CD19 CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

[0199] In some embodiments, the CD20 CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

[0200] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0201] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0202] In some embodiments, the engineered T cells are expanded from primary T cells or their progeny, or are derived from T cells differentiated from iPSCs or their progeny.

[0203] In some embodiments, the engineered T cells are differentiated cells derived from induced pluripotent stem cells or their progeny.

[0204] In some embodiments, the differentiated cells are T cells or natural killer (NK) cells.

[0205] In some embodiments, the engineered T cells are the progeny of a primary immune cell, optionally, the progeny of the primary immune cell is a T cell or an NK cell.

[0206] In some embodiments, the engineered T cells comprise reduced expression of beta-2-microglobulin (B2M) and / or MHC class II transactivator of transcription (CIITA) compared to intact or unmodified wild-type or control cells.

[0207] In some embodiments, the engineered T cells do not express B2M and / or CIITA.

[0208] In some embodiments, the engineered T cells comprise reduced expression of TCR-alpha and / or TCR-beta.

[0209] In some embodiments, the engineered T cells do not express TCR-alpha and / or TCR-beta.

[0210] In some embodiments, the one or more tolerogenic factors are selected from the group consisting of CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, MANF, and Serpinb9.

[0211] In some embodiments, the one or more tolerogenic factors comprises CD47.

[0212] In some embodiments, the CD19-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0213] In some embodiments, the CD19-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0214] In some embodiments, the CD20-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0215] In some embodiments, the CD20-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0216] In some embodiments, one or more of the first, second, and / or third exogenous polynucleotides or the bicistronic polynucleotide are inserted into the first, second, and / or third specific locus of at least one allele of the cell.

[0217] In some embodiments, the first, second, and / or third specific loci are selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0218] In some embodiments, the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus.

[0219] In some embodiments, the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

[0220] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0221] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using CRISPR / Cas gene editing.

[0222] In some embodiments, the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas2b.

[0223] In some embodiments, the CRISPR / Cas system is selected from the group consisting of: optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; optionally selected from the group consisting of Cas9, Csn2, and Cas4; optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and and Csx10, optionally Csf1, and optionally a Cas effector protein selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d), and optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0224] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0225] In some embodiments, the first, second and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cells using a lentiviral vector.

[0226] In some embodiments, the engineered T cells avoid NK cell-mediated cytotoxicity upon administration to a recipient patient.

[0227] In some embodiments, the engineered T cells are protected from cytolysis by mature NK cells upon administration to a recipient patient.

[0228] In some embodiments, the engineered T cells evade macrophage-mediated cytotoxicity, optionally including phagocytosis and / or reactive oxygen species.

[0229] In some embodiments, the engineered T cells do not induce an immune response against the cells upon administration to a recipient patient.

[0230] In some embodiments, the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, and pulmonary conditions.

[0231] In some embodiments, the administration is selected from the group consisting of intravenous injection, intramuscular injection, intravascular injection, and implantation.

[0232] In some embodiments, the engineered T cells are administered to the patient before, during, or after the initiation of a different therapeutic regimen.

[0233] In some embodiments, the different treatment regimen is selected from the group consisting of re-administration of the same or different cells, and pre-treatment, co-treatment, or post-treatment with an additional agent.

[0234] In some embodiments, the different cells are autologous T cells or NK cells, or CAR-T cells expressing a first CAR that is different from a second CAR expressed by the engineered CAR-T cell.

[0235] In some embodiments, the patient has been treated with an immunodepleting therapy prior to administering the engineered T cells.

[0236] In some embodiments, the immunodepletion therapy comprises administration of fludarabine and / or cyclophosphamide.

[0237] In some embodiments, the patient has previously received antibody therapy.

[0238] In some embodiments, the antibody therapy is rituximab.

[0239] In some embodiments, the immunodepletion therapy is about 1-50 mg / m 2 This involves an IV infusion of fludarabine for approximately 1 to 7 days.

[0240] In some embodiments, the immunodepletion therapy is administered at a dose of about 1, about 5, about 10, about 20, about 30, about 40, or about 50 mg / m 2 of fludarabine administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0241] In some embodiments, the immunodepletion therapy is about 30 mg / m 2 This involves an IV infusion of fludarabine for approximately four days.

[0242] In some embodiments, the immunodepletion therapy is about 100-1000 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately 1 to 7 days.

[0243] In some embodiments, the immunodepletion therapy is administered at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 mg / m 2of cyclophosphamide administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0244] In some embodiments, the immunodepletion therapy is about 500 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately two days.

[0245] In some embodiments, at least about 40×10 4 The engineered T cells are administered to the patient.

[0246] In some embodiments, at least about 40×10 5 The engineered T cells are administered to the patient.

[0247] In some embodiments, the engineered T cells persist in the subject for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0248] In some embodiments, the therapeutic effect of the engineered T cells lasts for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0249] In some embodiments, wild-type or control cells are the starting material.

[0250] In some embodiments, the unaltered or unmodified wild-type or control cells are starting T cells isolated from a donor.

[0251] In some embodiments, provided herein are methods of treating a patient with Epstein-Barr virus (EBV) infection, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen (HLA) molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0252] In some embodiments, provided herein are methods of treating a patient with EBV infection, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain with specificity for CD19 or CD22, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0253] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0254] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of beta-2-microglobulin (B2M) compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0255] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and MHC class II transcription activator (CIITA) relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0256] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain with specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0257] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0258] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0259] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, reduced expression of TCR-alpha (TRAC) and / or TCR-beta (TRB) compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or EBV antigen.

[0260] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0261] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0262] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0263] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0264] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0265] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising evaluating and / or diagnosing the patient for EBV infection and optionally multiple sclerosis, and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0266] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0267] In some embodiments, one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0268] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0269] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0270] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0271] In some embodiments, one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0272] In some embodiments, one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0273] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0274] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0275] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0276] In some embodiments, one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0277] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0278] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

[0279] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD20 CAR having the CDR sequence of SEQ ID NO: 37.

[0280] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV antigen CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172.

[0281] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequence of SEQ ID NO: 117.

[0282] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD22 CAR having the CDR sequence of SEQ ID NO: 45.

[0283] In some embodiments, provided herein are methods of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequences of SEQ ID NO: 117 and a CD22 CAR having the CDR sequences of SEQ ID NO: 45.

[0284] In some embodiments, provided herein is a method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134, or the one or more CARs have an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CARs have scFv sequences comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0285] In some embodiments, the methods further comprise assessing the patient for and / or diagnosing the patient with EBV infection and optionally multiple sclerosis prior to administering the population of engineered T cells to the patient.

[0286] In some embodiments, diagnosing comprises evaluating a patient for EBV infection.

[0287] In some embodiments, diagnosing comprises evaluating a patient for multiple sclerosis.

[0288] In some embodiments, treating prevents multiple sclerosis.

[0289] In some embodiments, the treatment treats multiple sclerosis.

[0290] In some embodiments, the EBV infected patient has been diagnosed with multiple sclerosis.

[0291] In some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis.

[0292] In some embodiments, the patient experiences remission of multiple sclerosis after administration of the engineered T cells.

[0293] In some embodiments, the EBV infected patient is undergoing treatment for EBV infection.

[0294] In some embodiments, the EBV infected patient has an active EBV infection.

[0295] In some embodiments, the EBV infected patient has an inactive EBV infection.

[0296] In some embodiments, the patient experiences a reduction in EBV infection following administration of the engineered T cells, optionally wherein the reduction in EBV infection is characterized by a reduction in viral load.

[0297] In some embodiments, the treatment prevents the transformation of an EBV infection from an inactive EBV infection to an active EBV infection.

[0298] In some embodiments, the method results in B cell depletion.

[0299] In some embodiments, the engineered T cells comprise one or more of 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, and an EBV antigen-specific CAR.

[0300] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain with specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0301] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0302] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0303] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0304] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0305] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0306] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0307] In some embodiments, one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0308] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0309] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0310] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0311] In some embodiments, one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0312] In some embodiments, one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0313] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0314] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0315] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0316] In some embodiments, one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0317] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0318] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

[0319] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0320] In some embodiments, the method further comprises evaluating the patient for and / or diagnosing the patient with an autoimmune disease.

[0321] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117 and a CD22 CAR having a CDR sequence of SEQ ID NO: 45, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0322] In some embodiments, the encoded CD19 CAR has the corresponding amino acid sequence set forth in SEQ ID NO: 117 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 SEQ ID NO: 117 and comprises the following components: CD8α signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8α hinge domain, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

[0323] In some embodiments, the encoded CD22 CAR has the corresponding amino acid sequence set forth in SEQ ID NO:45 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 SEQ ID NO:45.

[0324] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV antigen-specific CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172, and wherein the autoimmune disease is multiple sclerosis.

[0325] In some embodiments, provided herein is a method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0326] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0327] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0328] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0329] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0330] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0331] In some embodiments, provided herein are methods of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0332] In some embodiments, the methods further comprise assessing the patient for and / or diagnosing the patient with an autoimmune disease prior to administering the population of engineered T cells to the patient.

[0333] In some embodiments, the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, and pulmonary conditions.

[0334] In some embodiments, the patient is suspected of having or has been diagnosed with an EBV infection.

[0335] In some embodiments, the patient is suspected of having or has been diagnosed with multiple sclerosis.

[0336] In some embodiments, the method further comprises administering a second, third, fourth, fifth, or sixth dose of the engineered T cells to the patient.

[0337] In some embodiments, one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells are the same.

[0338] In some embodiments, the CAR is different in one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells.

[0339] In some embodiments, the CAR has an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy chain and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0340] In some embodiments, the CAR has the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, and 134.

[0341] 79. The method of any one of claims 1 to 78, wherein the engineered T cells comprise a CD19-specific CAR and a CD20-specific CAR.

[0342] In some embodiments, the CD19-specific CAR has the CDR sequences of SEQ ID NO: 117 and the CD22 CAR has the CDR sequences of SEQ ID NO: 45.

[0343] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

[0344] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

[0345] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

[0346] In some embodiments, the CD19 CAR T cells and the CD20 CAR T cells are administered simultaneously.

[0347] In some embodiments, the CD19 CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

[0348] In some embodiments, the CD19 CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

[0349] In some embodiments, the CD20 CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

[0350] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0351] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0352] In some embodiments, the engineered T cells comprise an EBV antigen-specific CAR and a CD20-specific CAR.

[0353] In some embodiments, the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

[0354] In some embodiments, the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

[0355] In some embodiments, the EBV antigen-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

[0356] In some embodiments, the EBV antigen CAR T cells and the CD20 CAR T cells are administered simultaneously.

[0357] In some embodiments, the EBV antigen CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

[0358] In some embodiments, the EBV antigen CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

[0359] In some embodiments, the CD20 CAR+ T cells are administered prior to administration of the EBV antigen CAR+ T cells.

[0360] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD20 CAR T cells alone.

[0361] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD20 CAR T cells alone.

[0362] In some embodiments, the engineered T cells comprise an EBV antigen-specific CAR and a CD19-specific CAR.

[0363] In some embodiments, the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bicistronic polynucleotide.

[0364] In some embodiments, the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bispecific CAR.

[0365] In some embodiments, the EBV antigen-specific CAR and the CD19-specific CAR are encoded by two separate polynucleotides.

[0366] In some embodiments, the EBV antigen CAR T cells and the CD19 CAR T cells are administered simultaneously.

[0367] In some embodiments, the EBV antigen CAR+ T cells and the CD19 CAR+ T cells are administered sequentially.

[0368] In some embodiments, the EBV antigen CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

[0369] In some embodiments, the CD19 CAR+ T cells are administered prior to administration of the EBV antigen CAR+ T cells.

[0370] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD19 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD19 CAR T cells alone.

[0371] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD19 CAR T cells is less than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD19 CAR T cells alone.

[0372] In some embodiments, the engineered T cells comprise an EBV antigen-specific CAR and a CD22-specific CAR.

[0373] In some embodiments, the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

[0374] In some embodiments, the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bispecific CAR.

[0375] In some embodiments, the EBV antigen-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

[0376] In some embodiments, the EBV antigen CAR T cells and the CD22 CAR T cells are administered simultaneously.

[0377] In some embodiments, the EBV antigen CAR+ T cells and the CD22 CAR+ T cells are administered sequentially.

[0378] In some embodiments, the EBV antigen CAR+ T cells are administered prior to administration of the CD22 CAR+ T cells.

[0379] In some embodiments, the CD22 CAR+ T cells are administered prior to administration of the EBV antigen CAR+ T cells.

[0380] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD22 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD22 CAR T cells alone.

[0381] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD22 CAR T cells is less than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD22 CAR T cells alone.

[0382] In some embodiments, the engineered T cells are primary T cells, expanded from primary T cells or their progeny, or derived from T cells differentiated from iPSCs or their progeny.

[0383] In some embodiments, the engineered T cells are differentiated cells derived from induced pluripotent stem cells or their progeny.

[0384] In some embodiments, the differentiated cells are T cells or natural killer (NK) cells.

[0385] In some embodiments, the engineered T cells are primary T cells or are the progeny of primary immune cells, optionally, the progeny of the primary immune cells are T cells or NK cells.

[0386] In some embodiments, the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules compared to intact or unmodified wild-type or control cells.

[0387] In some embodiments, the engineered T cells comprise reduced expression of one or more MHC HLA class II molecules compared to intact or unmodified wild-type or control cells.

[0388] In some embodiments, the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules and one or more MHC HLA class II molecules compared to intact or unmodified wild-type or control cells.

[0389] In some embodiments, the engineered T cells comprise reduced expression of B2M and / or CIITA compared to intact or unmodified wild-type or control cells.

[0390] In some embodiments, the engineered T cells do not express B2M and / or CIITA.

[0391] In some embodiments, the engineered T cells comprise reduced expression of TRAC and / or TRB.

[0392] In some embodiments, the engineered T cells do not express TRAC and / or TRB.

[0393] In some embodiments, the engineered T cells comprise reduced expression of TRAC.

[0394] In some embodiments, the engineered T cells do not express TRAC.

[0395] In some embodiments, the engineered T cells comprise reduced expression of TRB.

[0396] In some embodiments, the engineered T cells do not express TRB.

[0397] In some embodiments, the engineered T cells comprise reduced expression of TRAC and TRB.

[0398] In some embodiments, the one or more tolerogenic factors are selected from the group consisting of CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, and MANF, and Serpinb9; optionally, the one or more tolerogenic factors comprises CD47.

[0399] In some embodiments, the CD19-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0400] In some embodiments, the CD19-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0401] In some embodiments, the CD20-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0402] In some embodiments, the CD20-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0403] In some embodiments, one or more of the first, second, and / or third exogenous polynucleotides or the bicistronic polynucleotide are inserted into the first, second, and / or third specific locus of at least one allele of the cell.

[0404] In some embodiments, the first, second, and / or third specific loci are selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0405] In some embodiments, the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus.

[0406] In some embodiments, the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

[0407] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0408] In some embodiments, the gene therapy vector is a retrovirus or a fusosome.

[0409] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using CRISPR / Cas gene editing.

[0410] In some embodiments, the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0411] In some embodiments, the CRISPR / Cas system is selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally, Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); (f) optionally, a Cas effector protein selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0412] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0413] In some embodiments, the first, second and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cells using a lentiviral vector.

[0414] In some embodiments, the engineered T cells avoid NK cell-mediated cytotoxicity when administered to a patient.

[0415] In some embodiments, the engineered T cells are protected from cytolysis by mature NK cells upon administration to a patient.

[0416] In some embodiments, the engineered T cells evade macrophage-mediated cytotoxicity, optionally including phagocytosis and / or reactive oxygen species.

[0417] In some embodiments, the engineered T cells do not induce an immune response against the cells when administered to a patient.

[0418] In some embodiments, the administration is selected from the group consisting of intravenous injection, intramuscular injection, intravascular injection, and implantation.

[0419] In some embodiments, the engineered T cells are administered to the patient before, during, or after the initiation of a different therapeutic regimen.

[0420] In some embodiments, the different treatment regimen is selected from the group consisting of re-administration of the same or different cells, and pre-treatment, co-treatment, or post-treatment with an additional agent.

[0421] In some embodiments, the different cells are autologous T cells or NK cells, or CAR-T cells expressing a first CAR that is different from a second CAR expressed by the engineered CAR-T cell.

[0422] In some embodiments, the patient has been treated with an immunodepleting therapy prior to administering the engineered T cells.

[0423] In some embodiments, the immunodepletion therapy comprises administration of fludarabine and / or cyclophosphamide.

[0424] In some embodiments, the patient has previously received antibody therapy.

[0425] In some embodiments, the antibody therapy is rituximab.

[0426] In some embodiments, the immunodepletion therapy is about 1-50 mg / m 2 This involves an IV infusion of fludarabine for approximately 1 to 7 days.

[0427] In some embodiments, the immunodepletion therapy is administered at a dose of about 1, about 5, about 10, about 20, about 30, about 40, or about 50 mg / m2 of fludarabine administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0428] In some embodiments, the immunodepletion therapy is about 30 mg / m 2 This involves an IV infusion of fludarabine for approximately four days.

[0429] In some embodiments, the immunodepletion therapy is about 100-1000 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately 1 to 7 days.

[0430] In some embodiments, the immunodepletion therapy is administered at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 mg / m 2 of cyclophosphamide administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0431] In some embodiments, the immunodepletion therapy is about 500 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately two days.

[0432] In some embodiments, at least about 40×10 4 The engineered T cells are administered to the patient.

[0433] In some embodiments, at least about 40×10 5 The engineered T cells are administered to the patient.

[0434] In some embodiments, the engineered T cells persist in the subject for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0435] In some embodiments, the therapeutic effect of the engineered T cells lasts for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0436] In some embodiments, wild-type or control cells are the starting material.

[0437] In some embodiments, provided herein is the use of a population of engineered T cells for treating EBV infection in a patient suspected of having or diagnosed with EBV infection, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen (HLA) molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0438] In some embodiments, provided herein is the use of a population of engineered T cells for treating EBV infection in a patient suspected of having or diagnosed with EBV infection, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain with specificity for CD19 or CD22, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0439] In some embodiments, provided herein is the use of a population of engineered T cells for treating EBV infection in a patient suspected of having or diagnosed with EBV infection, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0440] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0441] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0442] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0443] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0444] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0445] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, reduced expression of TCR-alpha (TRAC) and / or TCR-beta (TRB) compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or EBV antigen.

[0446] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0447] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0448] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M relative to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0449] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0450] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0451] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0452] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

[0453] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0454] In some embodiments, one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0455] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0456] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0457] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0458] In some embodiments, one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0459] In some embodiments, one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0460] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0461] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0462] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0463] In some embodiments, one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0464] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0465] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

[0466] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD20 CAR having the CDR sequence of SEQ ID NO: 37.

[0467] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV antigen CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172.

[0468] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequence of SEQ ID NO: 117.

[0469] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD22 CAR having the CDR sequence of SEQ ID NO: 45.

[0470] In some embodiments, provided herein is the use of a population of engineered T cells for treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequence of SEQ ID NO: 117 and a CD22 CAR having the CDR sequence of SEQ ID NO: 45.

[0471] In some embodiments, provided herein is the use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134, or the one or more CARs have an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0472] In some embodiments, the use further comprises assessing the patient for EBV infection and optionally multiple sclerosis and / or diagnosing the patient with EBV infection and optionally multiple sclerosis prior to administering the population of engineered T cells to the patient.

[0473] In some embodiments, diagnosing comprises evaluating a patient for EBV infection.

[0474] In some embodiments, diagnosing comprises evaluating a patient for multiple sclerosis.

[0475] In some embodiments, treating prevents multiple sclerosis.

[0476] In some embodiments, the treatment treats multiple sclerosis.

[0477] In some embodiments, the EBV infected patient has been diagnosed with multiple sclerosis.

[0478] In some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis.

[0479] In some embodiments, the patient experiences remission of multiple sclerosis after administration of the engineered T cells.

[0480] In some embodiments, the EBV infected patient is undergoing treatment for EBV infection.

[0481] In some embodiments, the EBV infected patient has an active EBV infection.

[0482] In some embodiments, the EBV infected patient has an inactive EBV infection.

[0483] In some embodiments, the patient experiences a reduction in EBV infection following administration of the engineered T cells, optionally wherein the reduction in EBV infection is characterized by a reduction in viral load.

[0484] In some embodiments, the treatment prevents the transformation of an EBV infection from an inactive EBV infection to an active EBV infection.

[0485] In some embodiments, the use results in B cell depletion.

[0486] In some embodiments, the engineered T cells comprise one or more of 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, and an EBV antigen-specific CAR.

[0487] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain with specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0488] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0489] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0490] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0491] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0492] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0493] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0494] In some embodiments, one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0495] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0496] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0497] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0498] In some embodiments, one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0499] In some embodiments, one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0500] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0501] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0502] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0503] In some embodiments, one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0504] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0505] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

[0506] In some embodiments, provided herein is a method of using a population of engineered T cells to treat an autoimmune disease in a patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0507] In some embodiments, the use further comprises evaluating a patient for and / or diagnosing a patient with an autoimmune disease.

[0508] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having the CDR sequence of SEQ ID NO: 117 and a CD22 CAR having the CDR sequence of SEQ ID NO: 45, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

[0509] In some embodiments, the encoded CD19 CAR has the corresponding amino acid sequence set forth in SEQ ID NO: 117 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 SEQ ID NO: 117 and comprises the following components: CD8α signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8α hinge domain, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

[0510] In some embodiments, the encoded CD22 CAR has the corresponding amino acid sequence set forth in SEQ ID NO:45 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 SEQ ID NO:45.

[0511] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC compared to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV antigen-specific CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172, and the autoimmune disease is multiple sclerosis.

[0512] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0513] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0514] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0515] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0516] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0517] In some embodiments, provided herein is the use of a population of engineered T cells for treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0518] In some embodiments, provided herein is the use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules relative to intact or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

[0519] In some embodiments, the use further comprises assessing the patient for and / or diagnosing the patient with an autoimmune disease prior to administering the population of engineered T cells to the patient.

[0520] In some embodiments, the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, and pulmonary conditions.

[0521] In some embodiments, the patient is suspected of having or has been diagnosed with an EBV infection.

[0522] In some embodiments, the patient is suspected of having or has been diagnosed with multiple sclerosis.

[0523] In some embodiments, the use further comprises administering a second, third, fourth, fifth, or sixth dose of the engineered T cells to the patient.

[0524] In some embodiments, one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells are the same.

[0525] In some embodiments, the CAR is different in one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells.

[0526] In some embodiments, the CAR has an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising the heavy chain and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

[0527] In some embodiments, the CAR has the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, and 134.

[0528] In some embodiments, the engineered T cells comprise a CD19-specific CAR and a CD20-specific CAR.

[0529] In some embodiments, the CD19-specific CAR has the CDR sequences of SEQ ID NO: 117 and the CD22 CAR has the CDR sequences of SEQ ID NO: 45.

[0530] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

[0531] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

[0532] In some embodiments, the CD19-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

[0533] In some embodiments, the CD19 CAR T cells and the CD20 CAR T cells are administered simultaneously.

[0534] In some embodiments, the CD19 CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

[0535] In some embodiments, the CD19 CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

[0536] In some embodiments, the CD20 CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

[0537] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0538] In some embodiments, the number of cells administered as a therapeutically effective amount of CD19 and / or CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of CD19 CAR T cells or CD20 CAR T cells alone.

[0539] In some embodiments, the engineered T cells comprise an EBV antigen-specific CAR and a CD20-specific CAR.

[0540] In some embodiments, the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

[0541] In some embodiments, the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

[0542] In some embodiments, the EBV antigen-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

[0543] In some embodiments, the EBV antigen CAR T cells and the CD20 CAR T cells are administered simultaneously.

[0544] In some embodiments, the EBV antigen CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

[0545] In some embodiments, the EBV antigen CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

[0546] In some embodiments, the CD20 CAR+ T cells are administered prior to administration of the EBV antigen CAR+ T cells.

[0547] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD20 CAR T cells alone.

[0548] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD20 CAR T cells alone.

[0549] In some embodiments, the engineered T cells comprise an EBV antigen-specific CAR and a CD19-specific CAR.

[0550] In some embodiments, the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bicistronic polynucleotide.

[0551] In some embodiments, the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bispecific CAR.

[0552] In some embodiments, the EBV antigen-specific CAR and the CD19-specific CAR are encoded by two separate polynucleotides.

[0553] In some embodiments, the EBV antigen CAR T cells and the CD19 CAR T cells are administered simultaneously.

[0554] In some embodiments, the EBV antigen CAR+ T cells and the CD19 CAR+ T cells are administered sequentially.

[0555] In some embodiments, the EBV antigen CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

[0556] In some embodiments, the CD19 CAR+ T cells are administered prior to administration of the EBV antigen CAR+ T cells.

[0557] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD19 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD19 CAR T cells alone.

[0558] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD19 CAR T cells is less than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD19 CAR T cells alone.

[0559] In some embodiments, the engineered T cells comprise an EBV antigen-specific CAR and a CD22-specific CAR.

[0560] In some embodiments, the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

[0561] In some embodiments, the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bispecific CAR.

[0562] In some embodiments, the EBV antigen-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

[0563] In some embodiments, the EBV antigen CAR T cells and the CD22 CAR T cells are administered simultaneously.

[0564] In some embodiments, the EBV antigen CAR+ T cells and the CD22 CAR+ T cells are administered sequentially.

[0565] In some embodiments, the EBV antigen CAR+ T cells are administered prior to administration of the CD22 CAR+ T cells.

[0566] In some embodiments, the CD22 CAR+ T cells are administered prior to administration of the EBV antigen CAR+ T cells.

[0567] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD22 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD22 CAR T cells alone.

[0568] In some embodiments, the number of cells administered as a therapeutically effective amount of EBV antigen and / or CD22 CAR T cells is less than the number of cells administered as a therapeutically effective amount of EBV antigen CAR T cells or CD22 CAR T cells alone.

[0569] In some embodiments, the engineered T cells are primary T cells, expanded from primary T cells or their progeny, or derived from T cells differentiated from iPSCs or their progeny.

[0570] In some embodiments, the engineered T cells are differentiated cells derived from induced pluripotent stem cells or their progeny.

[0571] In some embodiments, the differentiated cells are T cells or natural killer (NK) cells.

[0572] In some embodiments, the engineered T cells are primary T cells and are the progeny of primary immune cells, optionally, the progeny of the primary immune cells are T cells or NK cells.

[0573] In some embodiments, the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules compared to intact or unmodified wild-type or control cells.

[0574] In some embodiments, the engineered T cells comprise reduced expression of one or more MHC HLA class II molecules compared to intact or unmodified wild-type or control cells.

[0575] In some embodiments, the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules and one or more MHC HLA class II molecules compared to intact or unmodified wild-type or control cells.

[0576] In some embodiments, the engineered T cells comprise reduced expression of B2M and / or CIITA compared to intact or unmodified wild-type or control cells.

[0577] In some embodiments, the engineered T cells do not express B2M and / or CIITA.

[0578] In some embodiments, the engineered T cells comprise reduced expression of TRAC and / or TRB.

[0579] In some embodiments, the engineered T cells do not express TRAC and / or TRB.

[0580] In some embodiments, the engineered T cells comprise reduced expression of TRAC.

[0581] In some embodiments, the engineered T cells do not express TRAC.

[0582] In some embodiments, the engineered T cells comprise reduced expression of TRB.

[0583] In some embodiments, the engineered T cells do not express TRB.

[0584] In some embodiments, the engineered T cells comprise reduced expression of TRAC and TRB.

[0585] In some embodiments, the one or more tolerogenic factors are selected from the group consisting of CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, MANF, and Serpinb9; optionally, the one or more tolerogenic factors comprises CD47.

[0586] In some embodiments, the CD19-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0587] In some embodiments, the CD19-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0588] In some embodiments, the CD20-specific CAR and one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

[0589] In some embodiments, the CD20-specific CAR and CD47 are encoded by a single bicistronic polynucleotide.

[0590] In some embodiments, one or more of the first, second, and / or third exogenous polynucleotides or the bicistronic polynucleotide are inserted into the first, second, and / or third specific locus of at least one allele of the cell.

[0591] In some embodiments, the first, second, and / or third specific loci are selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0592] In some embodiments, the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus.

[0593] In some embodiments, the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

[0594] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0595] In some embodiments, the gene therapy vector is a retrovirus or a fusosome.

[0596] In some embodiments, the first, second, and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cell using CRISPR / Cas gene editing.

[0597] In some embodiments, the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0598] In some embodiments, the CRISPR / Cas system is selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally, Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); (f) optionally, a Cas effector protein selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0599] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0600] In some embodiments, the first, second and / or third exogenous polynucleotide or bicistronic polynucleotide is introduced into the engineered T cells using a lentiviral vector.

[0601] In some embodiments, the engineered T cells avoid NK cell-mediated cytotoxicity when administered to a patient.

[0602] In some embodiments, the engineered T cells are protected from cytolysis by mature NK cells upon administration to a patient.

[0603] In some embodiments, the engineered T cells evade macrophage-mediated cytotoxicity, optionally including phagocytosis and / or reactive oxygen species.

[0604] In some embodiments, the engineered T cells do not induce an immune response against the cells when administered to a patient.

[0605] In some embodiments, the administration is selected from the group consisting of intravenous injection, intramuscular injection, intravascular injection, and implantation.

[0606] In some embodiments, the engineered T cells are administered to the patient before, during, or after the initiation of a different therapeutic regimen.

[0607] In some embodiments, the different treatment regimen is selected from the group consisting of re-administration of the same or different cells, and pre-treatment, co-treatment, or post-treatment with an additional agent.

[0608] In some embodiments, the different cells are autologous T cells or NK cells, or CAR-T cells expressing a first CAR that is different from a second CAR expressed by the engineered CAR-T cell.

[0609] In some embodiments, the patient has been treated with an immunodepleting therapy prior to administering the engineered T cells.

[0610] In some embodiments, the immunodepletion therapy comprises administration of fludarabine and / or cyclophosphamide.

[0611] In some embodiments, the patient has previously received antibody therapy.

[0612] In some embodiments, the antibody therapy is rituximab.

[0613] In some embodiments, the immunodepletion therapy is about 1-50 mg / m 2 This involves an IV infusion of fludarabine for approximately 1 to 7 days.

[0614] In some embodiments, the immunodepletion therapy is administered at a dose of about 1, about 5, about 10, about 20, about 30, about 40, or about 50 mg / m 2 of fludarabine administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0615] In some embodiments, the immunodepletion therapy is about 30 mg / m 2 This involves an IV infusion of fludarabine for approximately four days.

[0616] In some embodiments, the immunodepletion therapy is about 100-1000 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately 1 to 7 days.

[0617] In some embodiments, the immunodepletion therapy is administered at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 mg / m 2 of cyclophosphamide administered by IV infusion for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

[0618] In some embodiments, the immunodepletion therapy is about 500 mg / m 2 This involves an IV infusion of cyclophosphamide for approximately two days.

[0619] In some embodiments, at least about 40×10 4 The engineered T cells are administered to the patient.

[0620] In some embodiments, at least about 40×10 5 The engineered T cells are administered to the patient.

[0621] In some embodiments, the engineered T cells persist in the subject for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0622] In some embodiments, the therapeutic effect of the engineered T cells lasts for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

[0623] In some embodiments, wild-type or control cells are the starting material.

[0624] This disclosure is related to U.S. provisional patent applications filed on December 31, 2020 by Morrison and Foerester (Attorney Docket No. 112864-5057-PR) and January 11, 2021, having Attorney Docket No. 18615-30046.00, the contents of which are incorporated herein by reference in their entireties. A detailed description of engineered and / or hypoimmunogenic cells, methods for their production, and methods for their use can be found in U.S. Provisional Patent Application No. 63 / 065,342, filed August 13, 2020; WO2016 / 183041, filed May 9, 2015; WO2018 / 132783, filed January 14, 2018; WO2020 / 018615, filed July 17, 2019; WO2020 / 018620, filed July 17, 2019; and WO2020 / 168317, filed February 16, 2020, the disclosures of which, including the examples, sequence listings, and figures, are incorporated herein by reference in their entireties. DETAILED DESCRIPTION OF THE INVENTION

[0625] I. Introduction Described herein are engineered or modified immune evasive cells, including, but not limited to, human immune evasive cells, based in part on the hypoimmuned cell-editing platform described in WO2018132783 and PCT / US21 / 65157, filed December 23, 2021, each of which is incorporated herein by reference in its entirety. To overcome the problem of immune rejection by subjects of transplants derived from these primary cells and / or stem cells, the inventors have developed hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent cells, differentiated cells derived therefrom, and primary cells) that provide a viable source for any transplantable cell type, and are described herein. Such cells are protected from adaptive and / or innate immune rejection upon administration to a recipient subject. Advantageously, the cells disclosed herein are protected from adaptive and innate immune rejection upon administration to a recipient subject, and therefore are not rejected by the recipient subject's immune system, regardless of the subject's genetic makeup. In some embodiments, the engineered cells and / or hypoimmunogenic cells do not express major histocompatibility complex (MHC) class I and class II antigen molecules and / or T cell receptors. In certain embodiments, the engineered cells and / or hypoimmunogenic cells do not express MHC I and II antigen molecules and / or T cell receptors and overexpress CD47 protein. In certain embodiments, the engineered cells and / or hypoimmunogenic cells, e.g., engineered T cells and / or hypoimmunogenic T cells, do not express MHC I and II antigen molecules and / or T cell receptors, overexpress CD47 protein, and express an exogenous CAR.

[0626] In some embodiments, the hypoimmunogenic cells outlined herein are not subject to innate immune rejection. In some cases, hypoimmunogenic cells are not subject to NK cell-mediated lysis. In some cases, hypoimmunogenic cells are not subject to macrophage phagocytosis. In some embodiments, hypoimmunogenic cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that can be transplanted into a recipient subject with little or no need for immunosuppressive drugs. Such hypoimmunogenic cells retain cell-specific properties and characteristics after transplantation, such as pluripotency, and the ability to engraft and function similar to the corresponding native cells.

[0627] The technology disclosed herein utilizes expression of tolerogenic factors and modulation (e.g., reduction or elimination) of MHC I molecule, MHC II molecule, and / or TCR expression in human cells. In some embodiments, genome editing techniques utilizing rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of genes involved in immune response in cells (e.g., by deleting or inserting genomic DNA into genes involved in immune response to affect gene expression). In some embodiments, genome editing or other gene regulation techniques are used to insert tolerance-inducing (tolerogenic) factors into human cells, enabling the cells and their progeny (including any differentiated cells prepared from such cells) to evade immune recognition upon engraftment into a recipient subject. Thus, the cells described herein exhibit regulated expression of one or more genes and factors that affect MHC I molecule, MHC II molecule, and / or TCR expression and evade the immune system of a recipient subject.

[0628] Genome editing technology allows for double-stranded DNA breaks at desired gene loci. These controlled double-stranded breaks promote homologous recombination at specific gene loci. This process focuses on targeting specific sequences in nucleic acid molecules, such as chromosomes, with endonucleases, which recognize and bind to the sequences and induce double-stranded breaks in the nucleic acid molecules. The double-stranded breaks are repaired by either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0629] The practice of many embodiments will employ, unless otherwise indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology, which are within the skill of those in the art, many of which are described below by way of illustration, and such techniques are fully explained in the literature.For example, 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); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as Advances in Immunology.

[0630] II. Definition As described in this disclosure, the following terms are used and are defined as set forth below.

[0631] The term "antigen," as used herein, refers to a molecule capable of eliciting an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptidic and non-peptidic mimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, multicellular organisms such as viruses and viral extracts and parasites, and allergens. The term antigen broadly includes any type of molecule that is recognized as foreign by the host immune system.

[0632] The terms "autoimmune disease" or "autoimmune disorder" or "inflammatory disease" or "inflammatory disorder" refer to any disease or disorder in which a subject mounts an immune response against its own tissues and / or cells. Autoimmune disorders can affect almost every organ system in a subject (e.g., a human), including, but not limited to, diseases of the nervous system, gastrointestinal system, and endocrine system, 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.

[0633] In some embodiments, the autoimmune or inflammatory disorder includes arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic polyarthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis (plaque psoriasis, guttate psoriasis, pustular psoriasis, and psoriasis of the nails), atopy (including atopic diseases such as hay fever and Job's syndrome), dermatitis (including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, exfoliative psoriatic dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis), X-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria (including chronic allergic urticaria, chronic idiopathic urticaria, and chronic autoimmune urticaria), myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, and toxic epidermal necrolysis sclerosis (including systemic sclerosis), sclerosis (such as systemic sclerosis; multiple sclerosis (MS), MS associated with Epstein-Barr virus (EBV) infection, spino-ocular MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive relapsing MS, secondary progressive MS (SPMS), systemic progressive sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, and ataxic sclerosis), neuromyelitis optica spectrum disorder (NMO, also known as Devic's disease or Devic's syndrome), inflammatory bowel disease (IBD) including Crohn's disease; autoimmune-mediated gastrointestinal diseases; colon inflammation, e.g., ulcerative colitis, colonic ulcer, microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, and transmural colitis; and autoimmune inflammatory bowel disease), enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome (including adult or acute respiratory distress syndrome (ARDS)), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid of pregnancy, scrotal pruritus, autoimmune premature ovarian failure,sudden hearing loss due to autoimmune conditions, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis (such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis), glomerulonephritis (GN) with or without nephrotic syndrome (chronic or acute glomerulonephritis, primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN, or idiopathic GN) membranous nephropathy, membranous or membranoproliferative GN (MPGN) including types I and II, and rapidly progressive GN, or proliferative nephritis), autoimmune polyendocrine deficiency syndromes, balanitis including plasma cell focal balanitis, balanoposthitis, erythema annulare centrifugally, erythema dyspigmentosa perstans, erythema multiforme, granuloma annulare, lichen sclerosus and atrophicus, lichen simplex chronicus, lichen spinous, lichen planus, ichthyosis lamellar, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and reactions, eczema (allergic or atopic eczema, asteatotic eczema, dyshidrosis) eczema, and vesicular palmoplantar eczema), asthma (such as asthmatic bronchitis, bronchial asthma, and autoimmune asthma), conditions with T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood group during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus (lupus nephritis, lupus encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus), alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated lupus Type I diabetes, type II diabetes, and adult latent autoimmune diabetes (or type 1.5 diabetes), including lupus erythematosus; juvenile (type I) diabetes mellitus, including pediatric non-insulin-dependent diabetes mellitus (IDDM); adult-onset diabetes mellitus (type II diabetes); idiopathic diabetes; diabetes insipidus; diabetic retinopathy; diabetic nephropathy; and diabetic aortopathy; immune responses involving acute and delayed hypersensitivity mediated by cytokines and T lymphocytes; tuberculosis; sarcoidosis; granulomatous diseases (including lymphomatoid granulomatosis, Wegener's granulomatosis, or agranulocytopenia); vasculitis (vasculitis, large vessel vasculitis,Necrotizing vasculitis, such as polymyalgia rheumatica and giant cell (Takayasu's) arteritis, medium-sized vasculitis, Kawasaki disease and polyarteritis nodosa / periarteritis nodosa, microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis (such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs' positive anemia, Diamond-Blackfan anemia, hemolytic anemia, immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia perniciosa), Addison's disease, pure red cell anemia or pure red cell aplasia (PRCA); factor VIII deficiency; hemophilia A; autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte leakage, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes (e.g., secondary to sepsis, trauma, or hemorrhage), antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, and antiphospholipid antibodies syndrome, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous membrane pemphigoid, and pemphigus erythematous), autoimmune polyendocrinopathy, Reiter's disease or syndrome, burns, pre-eclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenia (e.g. occurring in patients with myocardial infarction), e.g. thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia, e.g. for example, idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP, acquired thrombocytopenic purpura, scleritis, e.g., idiopathic keratoscleritis, episcleritis, autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine gland diseases, e.g., thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis,autoimmune thyroid disease, idiopathic hypothyroidism, or Graves' disease), polyglandular syndromes, autoimmune polyglandular syndromes (or polyendocrinopathy syndromes), paraneoplastic syndromes including paraneoplastic neurological syndromes, e.g., Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, e.g., allergic encephalomyelitis (or encephalomyelitis allergic amyloidosis) and experimental allergic encephalomyelitis (EAE), myasthenia gravis, including thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, hepatitis, e.g., autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-graft) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatitis, transient acantholytic dermatosis, liver cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathy syndromes, celiac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis, e.g., refractory or relapsing or recurrent polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, shingles-associated pain, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis, e.g., monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathy, e.g., epilepsy, migraine, cardiac arrhythmias, muscle disorders, hearing loss, blindness, periodic paralysis, CNS channelopathy, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disorders,Fibromyalgia, polyendocrine deficiency, Schmidt's syndrome, adrenalitis, gastrotrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility (e.g., due to antisperm antibodies), mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortions, farmer's lung, erythema multiforme, postcardiotomy syndrome, post-myocardial infarction postcardiotomy syndrome, Cushing's syndrome, bird breeder's lung, allergic Granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases such as leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Kaplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Charmant's syndrome, Felty's syndrome, filariasis, chronic pulmonary fibro ... Cyclitis such as idiopathic cyclitis, metachronous cyclitis, iridocyclitis (acute or chronic), or Fuchs' cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune dysgonadism, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyroid Toxemia, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonia, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplanted organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthous stomatitis, arteriosclerotic disorders, spermatogenesis defects, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, phacosensitivity endophthalmitis, allergic enterocolitis, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hammann-Rich disease, sensorineural hearing loss,Paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, nonmalignant thymoma, vitiligo, toxic, Shock syndrome, food poisoning, conditions with T-cell infiltration, leukocyte adhesion deficiency, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases with leukocyte leakage, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyendocrine disease, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, polyglandular autoimmune syndrome type 1, adult-onset idiopathic hypoparathyroidism Epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal sinusitis, maxillary sinusitis, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary eosinophilic infiltrate, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonia, aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spinal cord injury adenitis, polyglandular autoimmune disease, sclerosing cholangitis, scleral, episcleral, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vascular ectasia, autoimmune disorders associated with connective tissue disease, rheumatism, nervous system diseases, lymphadenitis, decreased blood pressure response, vascular insufficiency, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases involving angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemia-reperfusion injury, reperfusion injury of myocardium or other tissues, phosphorus These include, but are not limited to, mycotic tracheobronchitis, inflammatory dermatitis, dermatitis with an acute inflammatory component, multiple organ failure, bullous disease, renal cortical necrosis, acute suppurative meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, narcolepsy, severe acute inflammation, chronic refractory inflammation, pyelitis, intra-arterial hyperplasia, peptic ulcer, valvular inflammation, emphysema, alopecia areata, adipose tissue inflammation / type II diabetes, obesity associated with adipose tissue inflammation / insulin resistance, endometriosis, and pulmonary hemosiderosis.

[0634] As used herein, "B cell depletion" refers to a reduction in B cell levels in an animal or human after cell or antibody therapy compared to B cell levels before treatment. B cell levels can be measured using well-known assays, such as obtaining a complete blood count or FACS analysis stained for known B cell markers. B cell depletion can be partial or complete. In one embodiment, CD20-expressing B cells are depleted by at least 25%. In one embodiment, CD19-expressing B cells are depleted by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In one embodiment, depletion of CD22-expressing B cells is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In one embodiment, depletion of CD19 and CD20-expressing B cells is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In one embodiment, depletion of B cells expressing CD19 and CD22 is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In one embodiment, depletion of B cells expressing CD20 and CD22 is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In some embodiments, depletion methods include those described in Ercoli, G. et al., Front Immunol. 11:611661 (2020), which is incorporated herein by reference in its entirety.

[0635] The term "chronic infectious disease" refers to a disease caused by an infectious agent in which the infection persists. Such diseases may include hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples may include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with cryptococcosis and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents may be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immune deficiency syndrome (AIDS).

[0636] As used herein, a "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 shown to result in improvement of at least one clinical endpoint relative to 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 result in statistically significant and meaningful efficacy for the treatment of the disease, disorder, or condition. In some embodiments, a clinically effective amount is also a therapeutically effective amount. In other embodiments, a clinically effective amount is not a therapeutically effective amount.

[0637] In some embodiments, the changes or modifications described herein (including, for example, genetic changes or modifications) result in reduced expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in reduced expression of a target or selected polypeptide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polypeptide sequence.

[0638] In additional or alternative embodiments, the present disclosure contemplates modifying target polynucleotide sequences by any method available to those skilled in the art, for example, using a TALEN system or RNA-guided transposase. Although examples of methods using CRISPR / Cas (e.g., Cas9 and Cas12a) and TALEN are described in detail herein, it should be understood that the present disclosure is not limited to the use of these methods / systems. Other methods known to those skilled in the art, for example, targeting B2M, can also be used herein to reduce or eliminate expression in target cells.

[0639] The term "equivalent," as used herein, refers to two or more factors, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between the two, and that reasonable conclusions can be drawn based on observed differences or similarities. Those skilled in the art will understand, in context, what level of identity is required in any given situation for two or more such factors, entities, circumstances, sets of conditions, etc. to be considered comparable. The control cell can be, for example, a comparable cell (e.g., the same cell type) that does not contain the relative alteration.

[0640] The terms "decreasing," "reduced," "reducing," and "decrease" are all used herein to generally mean a statistically significant decrease. However, for the avoidance of doubt, "decreasing," "reduced," "reducing," and "decrease" refer to a decrease of at least 10% compared to a reference level, e.g., a decrease 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% compared to a reference level, or a decrease of up to and including 100% (i.e., absent levels compared to a reference sample), or any decrease between 10-100%. In some embodiments, cells are engineered to reduce expression of one or more targets compared to unaltered or unmodified wild-type cells.

[0641] In some embodiments, the engineered and hypoimmunogenic cells described are derived from iPSCs or their progeny. As used herein, the term "derived from iPSCs or their progeny" encompasses the initial iPSC generated and any subsequent progeny thereof. As used herein, the term "progeny" encompasses, for example, first generation progeny, i.e., progeny directly derived, obtained, obtainable, or derivable from the initial iPSC, e.g., by conventional propagation methods. The term "progeny" encompasses further generations, e.g., second, third, fourth, fifth, sixth, seventh, or subsequent generations, i.e., generations of cells directly derived, obtained, obtainable, or derivable from the previous generation, e.g., by conventional propagation methods. The term "progeny" also encompasses modified cells resulting from modification or alteration of the initial iPSC or its progeny.

[0642] The term "donor subject" refers to an animal, e.g., a human, from which cells can be obtained. The terms "non-human animal" and "non-human mammal," used interchangeably herein, include 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. Preferably, however, the donor subject is a mammal, e.g., a human, or other mammal, e.g., a domestic mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc. A "donor subject" also refers to multiple donors, e.g., one or more human or non-human animals or non-human mammals.

[0643] The term "endogenous" refers to a reference molecule or polypeptide that is naturally present in a cell. Similarly, when used in reference to the expression of a coding nucleic acid, the term refers to the expression of a coding nucleic acid that is naturally contained in a cell and not exogenously introduced. Similarly, when used in reference to a promoter sequence, the term refers to a promoter sequence that is naturally contained in a cell and not exogenously introduced.

[0644] 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, a genetic change or modification that causes the engineered cell to differ from a wild-type cell.

[0645] As used herein, the term "exogenous" in the context of polynucleotide or polypeptide expression is intended to mean that the reference molecule or polypeptide is introduced into the cell of interest. The polypeptide can be introduced by introducing the encoding nucleic acid into the genetic material of the cell, for example, by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to introducing the encoding nucleic acid into the cell in an expressible form.

[0646] An "exogenous" molecule is a molecule, construct, factor, etc. 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 a cell" is determined relative to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of a neuron is an exogenous molecule relative to an adult neuronal cell. Exogenous molecules can include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0647] Exogenous molecules or factors can be, for example, small molecules produced by combinatorial chemistry processes, or macromolecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, and polysaccharides, any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include nucleic acids capable of forming duplexes as well as triplex-forming nucleic acids. See, for example, U.S. Patent 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.

[0648] The exogenous molecule or construct may be the same type of molecule as the endogenous molecule, such as an exogenous protein or nucleic acid. In such cases, the exogenous molecule is introduced into the cell at a concentration higher than the concentration of the endogenous molecule in the cell. In some cases, the exogenous nucleic acid may include an infectious viral genome, a plasmid or episome introduced into the cell, or a chromosome not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, biolistics, calcium phosphate precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0649] As used herein, "fusosome" includes a gene therapy vector comprising a retroviral vector pseudotyped with an engineered fusogen comprising a G protein modified to contain a targeting moiety and an F protein masked so that it does not recognize its cognate receptor. In some embodiments, the fusogen protein complex is derived from a paramyxovirus, optionally the paramyxovirus is Nipah virus. In some embodiments, the retroviral vector is a lentiviral vector.

[0650] For purposes of this disclosure, a "gene" includes all DNA regions that encode a gene product and that control the production of that gene product, whether or not such control sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and / or locus control regions.

[0651] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription 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 mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and / or glycosylation.

[0652] As used herein, the term "genetic modification" and its grammatical equivalents may refer to one or more changes in nucleic acids, for example, nucleic acids in the genome of an organism. For example, genetic modification may refer to the alteration, addition, and / or deletion of a gene or a portion of a gene or other nucleic acid sequence. Genetically modified cells may also refer to cells in which a gene or a portion of a gene has been added, deleted, and / or altered. Genetically modified cells may also refer to cells in which a nucleic acid sequence that is not a gene or a portion of a gene has been added. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms of a target gene or portion of a gene or nucleic acid sequence, and mechanisms that result in permanent knock-in, knock-down, or knock-out. Genetic modifications include, for example, both transient knock-in and mechanisms that result in permanent knock-in of a nucleic acid sequence. 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.

[0653] As used herein, the terms "grafting," "administering," "introducing," "implanting," and "transplanting," and grammatical variations thereof, are used interchangeably in the context of placing cells (e.g., cells described herein) into a subject by a method or route that results in the introduced cells localizing or at least partially localizing at a desired site or being introduced systemically (e.g., into the circulation). Cells may be directly implanted at the desired site or may be administered by any suitable route that results in delivery to the desired location in a subject, where at least some of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to as long as several days or years. In some embodiments, cells can also be administered (e.g., injected) to a location other than the desired site, such as intracerebrally or subcutaneously, e.g., in a capsule that maintains the implanted cells in the implanted location and prevents migration of the implanted cells.

[0654] The "HLA" or "human leukocyte antigen" or "HLA molecule" or "human leukocyte antigen molecule" complex is a gene complex that encodes human MHC proteins. These cell surface proteins, which comprise the HLA complex, are responsible for regulating immune responses to antigens. Humans have two MHC classes: class I and class II molecules, "HLA-I" and "HLA-II," or "HLA-I molecules" and "HLA-II molecules." HLA-I contains three proteins, HLA-A, HLA-B, and HLA-C, that present peptides derived from the inside of cells. Antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins associate with beta-2 microglobulin (B2M). HLA-II contains five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, that present extracellular antigens 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 intended to be limiting, as it depends on whether the gene is human (HLA) or mouse (MHC). Thus, when referring to mammalian cells, these terms can be used interchangeably herein.

[0655] The term "hypoimmunogenicity," as used herein to characterize cells, generally means that the cells are less susceptible to innate or adaptive immune rejection by a subject into which the cells are transplanted, e.g., the cells are less susceptible to allogeneic rejection by a subject into which the cells are transplanted. For example, such hypoimmunogenic cells are about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less susceptible to innate or adaptive immune rejection by a subject into which the cells are transplanted, compared to cells of the same type that do not contain the modification. In some embodiments, genome editing techniques are used to regulate the expression of MHC I and MHC II genes, thus contributing to the generation of hypoimmunogenic cells. In some embodiments, hypoimmunogenic cells avoid immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells generated from the hypoimmunogenic stem cells outlined herein avoid immune rejection when administered (e.g., transplanted or grafted) to an MHC-mismatched allogeneic recipient. In some embodiments, the hypoimmunogenic cells are protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection. Detailed descriptions of hypoimmunogenic cells, methods for their production, and methods for their use are found in WO2016183041, filed May 9, 2015; WO2018132783, filed January 14, 2018; WO2018176390, filed March 20, 2018; WO2020018615, filed July 17, 2019; WO2020018620, filed July 31, 2020; and WO2021222285, filed April 27, 2021, the disclosures of which, including the Examples, sequence listings and figures, are incorporated herein by reference in their entireties.

[0656] The low immunogenicity of cells can be determined by assessing the immunogenicity of the cells, such as their ability to induce adaptive and innate immune responses or to avoid eliciting such adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art. In some embodiments, immune response assays measure the effect of hypoimmunogenic cells on T cell proliferation, T cell activation, T cell killing, donor-specific antibody production, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, hypoimmunogenic cells and their derivatives exhibit reduced killing by T cells and / or NK cells when administered to a subject. In some cases, the cells and their derivatives exhibit reduced macrophage phagocytosis compared to unmodified or wild-type cells. In some embodiments, hypoimmunogenic cells induce a reduced or attenuated immune response in a recipient subject compared to corresponding unmodified wild-type cells. In some embodiments, hypoimmunogenic cells are non-immunogenic or unable to induce an immune response in a recipient subject.

[0657] 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 certain percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percent "identity" can be over a region of the sequences being compared, such as a functional domain, or over the entire length of the two sequences being compared. In sequence comparison, typically, one sequence serves as a reference sequence and is compared to a test sequence. When using a sequence comparison algorithm, the 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 of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0658] Optimal alignment of sequences for comparison can be performed, for example, 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 similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0659] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm 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.

[0660] As used herein, "immune signaling factor" refers to a molecule, protein, peptide, etc. that activates an immune signaling pathway, as the case may be.

[0661] As used herein, "immunosuppressive factors" or "immunoregulatory factors" or "tolerogenic factors" include hypoimmune factors, complement inhibitors, and other factors that modulate or influence the ability of cells to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment, which may be combined with additional genetic modifications.

[0662] The terms "increased," "increase," or "enhance," or "activate" are all used herein to generally mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," or "enhance," or "activate" mean an increase of at least 10% 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% compared to a reference level, or an increase of up to and including 100%, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase of 2-fold to more than 10-fold compared to a reference level. In some embodiments, the reference level, also referred to as the base level, is 0.

[0663] In some embodiments, the change is an indel. As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be understood by those skilled in the art, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the change is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one nucleotide. The gene editing (e.g., CRISPR / Cas) system of the present disclosure can be used to induce indels or point mutations of any length in a target polynucleotide sequence.

[0664] As used herein, "knockdown" refers to the reduction of the expression of target mRNA or corresponding target protein. Knockdown is generally reported relative to the level present after administration or expression of a non-control molecule (e.g., a non-target control shRNA, siRNA, or miRNA) that does not mediate the reduction of the RNA expression level. In some embodiments, the knockdown of target genes is achieved by conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, or conditional or inducible CRISPR interference (CRISPRi). In some embodiments, the knockdown of target genes is achieved by protein-based methods, such as conditional or inducible degron methods. In some embodiments, the knockdown of target genes is achieved by genetic modification, including shRNA, siRNA, miRNA, or by using a gene editing system (e.g., CRISPR / Cas).

[0665] Knockdown is generally assessed by measuring mRNA levels using quantitative polymerase chain reaction (qPCR) amplification or by measuring protein levels by Western blot or enzyme-linked immunosorbent assay (ELISA). Analyzing protein levels provides an assessment of both mRNA cleavage and translation inhibition. Additional techniques for measuring knockdown include RNA solution hybridization, nuclease protection, Northern hybridization, gene expression monitoring using microarrays, antibody binding, radioimmunoassay, and fluorescence-activated cell analysis. Based on the details described herein, those skilled in the art will easily understand how to use the gene editing system (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof.

[0666] As used herein, "knock-in" or "knock-in" refers to a genetic modification resulting from the insertion of a DNA sequence into a chromosomal locus in a host cell. This results in the initiation or increased levels of expression of the knocked-in gene, portion of a gene, or nucleic acid sequence insertion product, e.g., increased levels of RNA transcription and / or encoded protein. As will be understood by those skilled in the art, this can be accomplished in several ways, including inserting or adding one or more additional copies of a gene or portion thereof to the host cell, altering regulatory elements of an endogenous gene to increase protein expression, or inserting a specific nucleic acid sequence whose expression is desired. This may be achieved by modifying the promoter, adding a different promoter, adding enhancers, adding other regulatory elements, or modifying other gene expression sequences.

[0667] As used herein, "knockout" or "knock-out" includes deleting all or a portion of a target polynucleotide sequence to prevent translation or function of the target polynucleotide sequence. For example, knockout can be achieved by altering the target polynucleotide sequence by introducing an insertion or deletion ("indel") into the target polynucleotide sequence, including a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Based on the details provided herein, one of skill in the art will readily understand how to knock out a target polynucleotide sequence, or a portion thereof, using the disclosed gene editing systems (e.g., CRISPR / Cas).

[0668] In some embodiments, the genetic modification or alteration results in knockout or knockdown of a target polynucleotide sequence or a portion thereof. Knocking out a target polynucleotide sequence or a portion thereof using the gene editing system (e.g., CRISPR / Cas) of the present disclosure can be useful for various 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 the expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in a cell ex vivo and introducing the cell containing the knocked-out mutant allele into a subject), or for changing the genotype or phenotype of a cell.

[0669] "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 can also be complete (i.e., gene expression is completely inactivated or activated to above wild-type levels), or it can be partial (gene expression is partially reduced or partially activated to a fraction of wild-type levels).

[0670] In additional or alternative aspects, the present disclosure contemplates modifying a target polynucleotide sequence using any method available to one of skill in the art, for example, using a nuclease system, such as a TAL effector nuclease (TALEN) or zinc finger nuclease (ZFN) system. While exemplary methods utilizing CRISPR / Cas (e.g., Cas9 and Cas12a) and TALENs are detailed herein, it should be understood that the present disclosure is not limited to the use of these methods / systems. Other targeting methods known to those of skill in the art can be utilized herein to reduce or eliminate expression in target cells. The methods provided herein can be used to modify a target polynucleotide sequence within a cell. The present disclosure contemplates modifying a target polynucleotide sequence within a cell for any purpose. In some embodiments, a target polynucleotide sequence within a cell is modified to produce a mutant cell. As used herein, a "mutant cell" refers to a cell whose genotype differs from its original genotype due to a mutation. In some cases, a "mutant cell" exhibits a mutant phenotype, for example, when a normally functioning gene is modified using a gene editing system (e.g., a CRISPR / Cas) system of the present disclosure. In other cases, a "mutant cell" exhibits a wild-type phenotype, for example, when a gene editing system (e.g., a CRISPR / Cas) system of the present disclosure is used to correct the mutant genotype. In some embodiments, a target polynucleotide sequence in a cell is modified to correct or repair a genetic mutation (e.g., to return the cell to a normal phenotype). In some embodiments, a target polynucleotide sequence in a cell is modified to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0671] As used herein, the term "native cell" refers to a cell that has not been otherwise modified (e.g., engineered). In some embodiments, a native cell is a naturally occurring wild-type or control cell.

[0672] The terms "operably linked" or "operably linked" are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) that allow for the possibility that both components function normally and that at least one of the components may mediate a function on at least one of the other components. By way of example, a transcription control sequence, such as a promoter, is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcription factors. A transcription control sequence is generally operably linked to a coding sequence in cis, but need not be directly adjacent to it. For example, an enhancer is a transcription control sequence that is operably linked to a coding sequence, even if they are not contiguous.

[0673] As used herein, "pluripotent stem cells" have the potential to differentiate into any of three germ layers: endoderm (e.g., stomach wall, digestive tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epithelial tissue and nervous system tissue). The term "pluripotent stem cells," as used herein, also encompasses "induced pluripotent stem cells," or "iPSCs," or a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, pluripotent stem cells are produced or generated from cells that are not pluripotent. In other words, pluripotent stem cells can be the direct or indirect progeny of a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be generated by inducing the expression of specific regulatory genes or by exogenously applying specific proteins. Methods for deriving iPS cells are known in the art and are described in detail below. (See, e.g., Zhou et al., Stem Cells 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol. 26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009), each of which is incorporated herein by reference in its entirety.) The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, "hiPSCs" refers to human induced pluripotent stem cells. In some embodiments, "pluripotent stem cells" as used herein also encompass mesenchymal stem cells (MSCs) and / or embryonic stem cells (ESCs).

[0674] 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 instances, a promoter sequence includes a transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. In some embodiments, a promoter sequence includes not only the transcription initiation site but also a protein binding domain responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes.

[0675] In some embodiments, the described engineered and hypoimmunogenic cells are expanded from primary T cells or their progeny. As used herein, the term "expanded from primary T cells or their progeny" includes primary T cells isolated from a donor subject and any subsequent progeny thereof. As used herein, the term "progeny" includes, for example, first generation progeny, i.e., progeny directly derived, obtained, obtainable, or derivable from the initial primary T cell, e.g., by conventional propagation methods. The term "progeny" includes further generations, e.g., second, third, fourth, fifth, sixth, seventh, or subsequent generations, i.e., generations of cells directly derived, obtained, obtainable, or derivable from the previous generation, e.g., by conventional propagation methods. The term "progeny" also includes modified cells resulting from the modification or alteration of primary T cells or their progeny.

[0676] The term "recipient patient" refers to an animal, e.g., a human, to which treatment, including prophylactic treatment, with the cells described herein is provided. In the case of treatment of an infection, condition, or disease state specific to a particular animal, such as a human patient, the term patient refers to that particular animal. The term "recipient patient" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the recipient patient is a mammal, e.g., a human, or other mammal, e.g., a domestic mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc.

[0677] As used herein, the terms "control sequence," "control element," and "regulatory element" are used interchangeably and refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' untranslated sequences) of an expressed polynucleotide target. Control sequences influence, for example, but not limited to, the timing of transcription, the amount or level of transcription, RNA processing or stability, and / or translation of associated structural nucleotide sequences. Control sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like. Since in most cases the exact boundaries of control sequences have not been completely defined, it is recognized that nucleotide sequences of different lengths can have identical control or promoter activity.

[0678] As used herein, a "safe harbor locus" refers to a genetic locus that allows a newly inserted genetic element to function as expected and allows expression of a transgene or exogenous gene in a manner that does not cause changes in the host genome that pose a risk to the host cell. Exemplary "safe harbor" genetic loci include, but are not limited to, the CCR5 gene, the PPP1R12C (also known as AAVS1) gene, the CLYBL gene, and / or the Rosa gene (e.g., ROSA26).

[0679] As used herein, a "target locus" refers to a locus that allows expression of a transgene or exogenous gene. Exemplary "target loci" include, but are not limited to, the CXCR4 gene, albumin gene, SHS231 locus, F3 gene (also known as CD142), MICA gene, MICB gene, LRP1 gene (also known as CD91), HMGB1 gene, ABO gene, RHD gene, FUT1 gene, and / or KDM5D gene (also known as HY). An exogenous polynucleotide encoding an exogenous gene may be inserted into the CDS region of B2M, CIITA, TRAC, TRBC, CCR5, F3 (i.e., CD142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D (i.e., HY), PDGFRa, OLIG2, and / or GFAP. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS1) or CCR5. An exogenous polynucleotide encoding an exogenous gene may be inserted into exon 1, 2, or 3 of CCR5. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 2 of CLYBL. An exogenous polynucleotide encoding an exogenous gene may be inserted into a 500 bp window in Ch-4:58,976,613 (i.e., SHS231). An exogenous polynucleotide encoding an exogenous gene may be inserted into any suitable region of the aforementioned safe harbor or target locus that allows exogenous expression, including, for example, an intron, exon, or coding sequence region in the safe harbor or target locus.

[0680] As used herein, "target" refers to a gene, part of a gene, part of a genome, or protein that is subject to controllably reduced expression by the methods described herein.

[0681] As used herein, a "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 the symptoms or side effects of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is also a clinically effective amount. In other embodiments, a therapeutically effective amount is not a clinically effective amount.

[0682] As used herein, the terms "treating" and "treatment" include administering a therapeutically or clinically effective amount of a cell described herein to a subject so that the subject experiences at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired therapeutic or clinical result. For purposes of the present technology, a beneficial or desired therapeutic or clinical result includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete). Treatment may refer to a prolongation of survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art will recognize that treatment may improve the disease state, but may not completely cure 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.

[0683] For purposes of the present technology, beneficial or desired therapeutic or clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete).

[0684] A "vector" or "construct" is capable of transferring a gene sequence into a target cell. Typically, the terms "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct that can induce expression of a gene of interest and transfer a gene sequence into a target cell. Thus, the term includes cloning and expression vehicles as well as integrating vectors. Methods for introducing vectors or constructs into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing 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.

[0685] In some embodiments, cells are engineered to reduce or increase expression of one or more targets relative to unaltered or unmodified wild-type cells. In some embodiments, cells are engineered to constitutively reduce or increase expression of one or more targets relative to unaltered or unmodified wild-type cells. In some embodiments, cells are engineered to controllably reduce or increase expression of one or more targets relative to unaltered or unmodified wild-type cells. In some embodiments, cells comprise increased expression of CD47 relative to wild-type cells or control cells of the same cell type. "Wild-type" or "wt" or "control" in the context of cells refers to 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, when used herein in the context of engineered cells, "wild-type" or "control" also refers to engineered cells that may contain nucleic acid changes that reduce expression of one or more MHC class I and / or class II molecules and / or T cell receptors, but that have not undergone a gene editing procedure that results in overexpression of CD47 protein. For example, as used herein, "wild-type" or "control" refers to engineered cells that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild-type" or "control" refers to engineered cells that contain reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. As used herein, "wild-type" or "control" also refers to engineered cells that may contain nucleic acid alterations that result in overexpression of CD47 protein but have not undergone a gene editing procedure that results in reduced expression of one or more MHC class I molecules and / or class II molecules and / or T cell receptors. In the context of iPSCs or their progeny, "wild-type" or "control" also refers to iPSCs or their progeny that may contain nucleic acid alterations that result in pluripotency but have not undergone a gene editing procedure of the present disclosure that achieves reduced expression of one or more MHC class I molecules and / or class II molecules and / or T cell receptors and / or overexpression of CD47 protein.For example, as used herein, "wild-type" or "control" refers to iPSCs or their progeny that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild-type" or "control" refers to iPSCs or their progeny that contain reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. In the context of primary T cells or their progeny, "wild-type" or "control" also refers to primary T cells or their progeny that may contain nucleic acid alterations that result in reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors, but that have not undergone a gene editing procedure that results in overexpression of CD47 protein. For example, as used herein, "wild-type" or "control" refers to primary T cells or their progeny that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild-type" or "control" refers to a primary T cell or its progeny that contains reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. In the context of a primary T cell or its progeny, "wild-type" or "control" also refers to a primary T cell or its progeny that may contain a nucleic acid alteration that results in overexpression of CD47 protein, but that has not undergone a gene editing procedure that results in reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors. In some embodiments, the cells are engineered to controllably reduce or increase expression of one or more targets compared to cells of the same cell type that do not contain the alteration. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0686] It should be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "only," "only," and the like, in connection with the recitation of claim elements, or the use of a "negative" limitation. It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has distinct elements and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the present disclosure. Any recited method can 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 can be used in the practice or testing of the present disclosure, representative exemplary methods and materials are described below.

[0687] Unless otherwise defined, 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. When a range of values ​​is described, it is understood that each value between the upper and lower limit of that range, to the tenth of the unit of the lower limit unless expressly stated otherwise, and any other stated or intervening value within that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included in the narrower range, and are also encompassed within the disclosure, if any specific limit in the stated range is excluded. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Certain ranges are presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the number it precedes, as well as for a number that is near or approximately the number it precedes. When determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, provides a substantial equivalent value to 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.

[0688] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and does not constitute an admission that the technology described herein is not entitled to antedate such publication by prior art. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0689] Before further describing the present invention, it is to be understood that the present technology is not limited to particular embodiments described, which 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, as the scope of the disclosure will be limited only by the appended claims. It is also to be understood that the headings used herein are not limiting, but are merely intended to provide orientation to the reader, but that the subject matter applies generally to the technology disclosed herein.

[0690] III. Detailed Description A. Low immunogenic cells In some embodiments, the present disclosure is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (such as, but not limited to, T cells and NK cells), and primary cells (such as, but not limited to, primary T cells and primary NK cells). In some embodiments, pluripotent stem cells, differentiated cells derived from such cells, such as T cells and NK cells, and primary cells, such as primary T cells and primary NK cells, are engineered to have reduced or no expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and in some cases, to have reduced or no expression of the T cell receptor (TCR) complex. In some embodiments, hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR) and have reduced or no expression of the T cell receptor (TCR) complex, in addition to reduced or no expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules. In some embodiments, the CAR comprises an antigen binding domain that binds to any one selected from the group consisting of CD19, CD22, CD20, BCMA, anEBV antigen, CD27, CD30, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, and gH / gL. In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the CAR is a CD20-specific CAR. In some cases, the CAR is a BCMA-specific CAR. In some embodiments, the CAR is an EBV antigen-specific CAR. In some embodiments, the CAR is a CD27-specific CAR. In some embodiments, the CAR is a CD30-specific CAR. In some embodiments, the CAR is an EBNA1-specific CAR. In some embodiments, the CAR is an EBNA3A-specific CAR. In some embodiments, the CAR is a BRLF1-specific CAR. In some embodiments, the CAR is a BALF4-specific CAR. In some embodiments, the CAR is an EBNA3C-specific CAR.In some embodiments, the CAR is an LMP1-specific CAR. In some embodiments, the CAR is an LMP2-specific CAR. In some embodiments, the CAR is an LMP2A-specific CAR. In some embodiments, the CAR is an LMP2B-specific CAR. In some embodiments, the CAR is a BZLF1-specific CAR. In some embodiments, the CAR is a BMLF1-specific CAR. In some embodiments, the CAR is a gp350-specific CAR. In some embodiments, the CAR is a gH / gL-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD20 bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR. In some embodiments, the bispecific CAR is an EBV antigen / CD20 bispecific CAR. In some embodiments, the bispecific CAR is an EBV antigen / CD19 bispecific CAR. In some embodiments, the bispecific CAR is an EBV antigen / CD22 bispecific CAR. In some embodiments, the described cells express a CD19-specific CAR and a different CAR, such as, but not limited to, a CD20-specific CAR, a BCMA-specific CAR, an EBV antigen-specific CAR, a CD27-specific CAR, a CD30-specific CAR, an EBNA1-specific CAR, an EBNA3A-specific CAR, a BRLF1-specific CAR, a BALF4-specific CAR, an EBNA3C-specific CAR, an LMP1-specific CAR, an LMP2-specific CAR, an LMP2A-specific CAR, an LMP2B-specific CAR, a BZLF1-specific CAR, a BMLF1-specific CAR, a gp350-specific CAR, and a gH / gL-specific CAR.In some embodiments, the described cells express a CD20-specific CAR and a different CAR, such as, but not limited to, a CD19-specific CAR, a BCMA-specific CAR, anEBV antigen-specific CAR, a CD27-specific CAR, a CD30-specific CAR, an EBNA1-specific CAR, an EBNA3A-specific CAR, a BRLF1-specific CAR, a BALF4-specific CAR, an EBNA3C-specific CAR, an LMP1-specific CAR, an LMP2-specific CAR, an LMP2A-specific CAR, an LMP2B-specific CAR, a BZLF1-specific CAR, a BMLF1-specific CAR, a gp350-specific CAR, and a gH / gL-specific CAR. In some embodiments, the described cells express an EBV antigen-specific CAR and a different CAR, such as, but not limited to, a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, a BCMA-specific CAR, an EBV antigen-specific CAR, a CD27-specific CAR, a CD30-specific CAR, an EBNA1-specific CAR, an EBNA3A-specific CAR, a BRLF1-specific CAR, a BALF4-specific CAR, an EBNA3C-specific CAR, an LMP1-specific CAR, an LMP2-specific CAR, an LMP2A-specific CAR, an LMP2B-specific CAR, a BZLF1-specific CAR, a BMLF1-specific CAR, a gp350-specific CAR, and a gH / gL-specific CAR. In some embodiments, the described cells express a CD22-specific CAR and a different CAR, for example, but not limited to, a CD19-specific CAR, a BCMA-specific CAR, an EBV antigen-specific CAR, a CD27-specific CAR, a CD30-specific CAR, an EBNA1-specific CAR, an EBNA3A-specific CAR, a BRLF1-specific CAR, a BALF4-specific CAR, an EBNA3C-specific CAR, an LMP1-specific CAR, an LMP2-specific CAR, an LMP2A-specific CAR, an LMP2B-specific CAR, a BZLF1-specific CAR, a BMLF1-specific CAR, a gp350-specific CAR, and a gH / gL-specific CAR. In some embodiments, the described cells express a BCMA-specific CAR and a different CAR, for example, but not limited to, a CD20-specific CAR, and a CD19-specific CAR.In some embodiments, the described cells express a BCMA-specific CAR and a different CAR, for example, but not limited to, a CD22-specific CAR and a CD19-specific CAR. In some embodiments, the described cells express a BCMA-specific CAR and a different CAR, for example, but not limited to, a CD20-specific CAR, a CD19-specific CAR, a CD22-specific CAR, an EBV antigen-specific CAR, a CD27-specific CAR, a CD30-specific CAR, an EBNA1-specific CAR, an EBNA3A-specific CAR, a BRLF1-specific CAR, a BALF4-specific CAR, an EBNA3C-specific CAR, an LMP1-specific CAR, an LMP2-specific CAR, an LMP2A-specific CAR, an LMP2B-specific CAR, a BZLF1-specific CAR, a BMLF1-specific CAR, a gp350-specific CAR, and a gH / gL-specific CAR. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to alter the expression of one or more genes to generate an engineered cell.

[0691] In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR) and comprise a genomic modification in the B2M gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and comprise a genomic modification in the CIITA gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR and comprise a genomic modification in the TRAC gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR and comprise a genomic modification in the TRB gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR and comprise one or more genomic modifications selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. In some embodiments, engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and CAR and contain genomic modifications of the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the cells also express CAR. - / - , C.I.T.A. - / - ,TRAC - / - , CD47tg cells. In some embodiments, engineered T cells and / or hypoimmune (HIP) T cells are generated by differentiating induced pluripotent stem cells, e.g., engineered induced pluripotent stem cells and / or hypoimmunogenic induced pluripotent stem cells. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0692] In some embodiments, engineered and / or hypoimmune (HIP) T cells and primary T cells are B2M T cells that also express a CAR. - / - , C.I.T.A. - / - , TRB- / - In some embodiments, the cells are B2M, CD47tg cells that also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , TRB - / - In certain embodiments, the cells are B2M, CD47tg cells that also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In certain embodiments, the cells are B2M, CD47tg cells that also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In certain embodiments, the cells are B2M, CD47tg cells that also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル, CD47tg cells. In some embodiments, the engineered or modified cells described are pluripotent stem cells, induced pluripotent stem cells, NK cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, T cells and induced pluripotent stem cells differentiated from such pluripotent stem cells, or primary T cells. 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, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue-resident memory (Trm) cells, virtual memory T cells, innate immune memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cell. In some embodiments, the primary T cells are selected from the group including cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor-infiltrating lymphocytes, and combinations thereof. Non-limiting examples of NK cells and primary NK cells include immature NK cells and mature NK cells. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to alter the expression of one or more genes to generate engineered cells.

[0693] In some embodiments, the primary T cells are from a pool of primary T cells derived from one or more donor subjects different from the recipient subject (e.g., the patient to whom the cells are administered). The primary T cells can be obtained and pooled together from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects. The primary T cells can be obtained and pooled together 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. In some embodiments, the primary T cells are harvested from one or more individuals, and in some cases, the primary T cells or pool of primary T cells are cultured in vitro. In some embodiments, the primary T cells or pool of primary T cells are engineered to exogenously express CD47 and cultured in vitro.

[0694] In certain embodiments, primary T cells or pools of primary T cells are engineered to express a chimeric antigen receptor (CAR). CARs can be any known to those skilled in the art. Useful CARs include those that bind to an antigen selected from the group consisting of CD19, CD20, CD22, BCMA, anEBV antigen, CD27, CD30, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, and gH / gL. Optionally, the CAR is the same as or equivalent to those used in FDA-approved CAR-T cell therapies, such as, but not limited to, those used in tisagenlecleucel and axicabtageneciloreucel, or others under investigation in clinical trials.

[0695] In some embodiments, the primary T cells or pool of primary T cells are engineered to exhibit reduced expression of an endogenous T cell receptor compared to unmodified primary T cells. In certain embodiments, the primary T cells or pool of primary T cells are engineered to exhibit reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1 compared to unmodified primary T cells. Methods for genetically modifying cells, including T cells, are described in detail, for example, in WO2020 / 018620 and WO2016 / 183041, the disclosures of which, including tables, appendices, sequence listings, and figures, are incorporated herein by reference in their entirety.

[0696] In some embodiments, the CAR-T cells comprise a CAR selected from the group comprising: (a) a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR.

[0697] In some embodiments, the 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 CD19 antigen binding domain. In some embodiments, the CAR comprises an EBV antigen binding domain. In some embodiments, the CAR comprises a CD27 binding domain. In some embodiments, the CAR comprises a CD30 binding domain. In some embodiments, the CAR comprises an EBNA1 binding domain. In some embodiments, the CAR comprises an EBNA3A binding domain. In some embodiments, the CAR comprises a BRLF1 binding domain. In some embodiments, the CAR comprises a BALF4 binding domain. In some embodiments, the CAR comprises an EBNA3C binding domain. In some embodiments, the CAR comprises an LMP1 binding domain. In some embodiments, the CAR comprises an LMP2 binding domain. In some embodiments, the CAR comprises an LMP2A binding domain. In some embodiments, the CAR comprises an LMP2B binding domain. In some embodiments, the CAR comprises a BZLF1 binding domain. In some embodiments, the CAR comprises a BMLF1 binding domain. In some embodiments, the CAR comprises a gp350 binding domain. In some embodiments, the CAR comprises a gH / gL binding domain. In some embodiments, the CAR comprises a CD28 or CD8α transmembrane domain. In some embodiments, the CAR comprises a CD8α signal peptide. In some embodiments, the CAR comprises the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the antigen binding domain of the CAR is selected from the group including, but not limited to: (a) an antigen binding domain that targets an antigen characteristic of neoplastic cells, (b) an antigen binding domain that targets an antigen characteristic of T cells, (c) an antigen binding domain that targets an antigen characteristic of an autoimmune disease / disorder and / or an inflammatory disease / disorder, (d) an antigen binding domain that targets an antigen characteristic of senescent cells, (e) an antigen binding domain that targets an antigen characteristic of an infectious disease, and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

[0698] In some embodiments, the CAR further comprises one or more linkers. The scFv format generally comprises two variable domains linked by a flexible peptide sequence or "linker" in either a VH-linker-VL or VL-linker-VH orientation. Any suitable linker known to those skilled in the art in light of this specification can be used in a CAR. Examples of suitable linkers include, but are not limited to, GS-based linker sequences and the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the linker is a GS or gly-ser linker. An exemplary gly-ser polypeptide linker has the amino acid sequence Ser (Gly4Ser) n , and (Gly4Ser) n and / or (Gly4Ser3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3. In some embodiments, n=4, i.e., Ser(Gly4Ser)4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser) nIn some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In other embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly3Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly3Ser)n. n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In other embodiments, n=5. In yet other embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly4Ser3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly3Ser) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In other embodiments, n=5. In yet other embodiments, n=6.

[0699] In some embodiments, the antigen-binding domain is selected from the group including an antibody, an antigen-binding portion thereof, an scFv, and a Fab. In some embodiments, the antigen-binding domain binds to CD19, CD20, CD22, BCMA, an EBV antigen, CD27, CD30, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, or gH / gL. In some embodiments, the antigen-binding domain is an anti-CD19 scFv, such as, but not limited to, FMC63. In some embodiments, the antigen-binding domain is an anti-CD20 scFv. In some embodiments, the antigen-binding domain is an anti-CD22 scFv. In some embodiments, the antigen-binding domain is an anti-BCMA scFv. In some embodiments, the antigen-binding domain is an anti-EBV antigen scFv. In some embodiments, the antigen-binding domain is an anti-CD27 scFv. In some embodiments, the antigen binding domain is an anti-CD30 scFv. In some embodiments, the antigen binding domain is an anti-EBNA1 scFv. In some embodiments, the antigen binding domain is an anti-EBNA3A scFv. In some embodiments, the antigen binding domain is an anti-BRLF1 scFv. In some embodiments, the antigen binding domain is an anti-BALF4 scFv. In some embodiments, the antigen binding domain is an anti-EBNA3C scFv. In some embodiments, the antigen binding domain is an anti-LMP1 scFv. In some embodiments, the antigen binding domain is an anti-LMP2 scFv. In some embodiments, the antigen binding domain is an anti-LMP2A scFv. In some embodiments, the antigen binding domain is an anti-LMP2B scFv. In some embodiments, the antigen binding domain is an anti-BZLF1 scFv. In some embodiments, the antigen binding domain is an anti-BMLF1 scFv. In some embodiments, the antigen binding domain is an anti-gp350 scFv. In some embodiments, the antigen-binding domain is an anti-gH / gL scFv.

[0700] In some embodiments, the transmembrane domain comprises one selected from the group comprising the transmembrane region of TCRα, TCRβ, TCRζ, CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0701] In some embodiments, the signaling domain(s) of the CAR comprise a costimulatory domain(s). For example, a single signaling domain can contain a single costimulatory domain. Alternatively, a signaling domain can contain one or more costimulatory domains. In certain embodiments, a signaling domain comprises a costimulatory domain. In other embodiments, a signaling domain comprises a costimulatory domain. Optionally, when a CAR comprises two or more costimulatory domains, the two costimulatory domains are not the same. In some embodiments, a costimulatory domain comprises two costimulatory domains that are not the same. In some embodiments, a costimulatory domain enhances cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence. In some embodiments, a costimulatory domain enhances cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence.

[0702] As described herein, a fourth-generation CAR may contain an antigen-binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful CAR signaling. In some cases, the cytokine gene is an endogenous or exogenous cytokine gene of the low-immunogenic cell. Optionally, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group including IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and functional fragments thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful CAR signaling comprises a transcription factor or a functional domain or fragment thereof.

[0703] In some embodiments, the CAR comprises a CD3 zeta (CD3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In other embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain o...

Claims

1. 1. A method of treating a patient with Epstein-Barr Virus (EBV) infection comprising administering to the patient a population of engineered T cells, the engineered T cells comprising reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen (HLA) molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), the one or more CARs comprising an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

2. 1. A method of treating a patient with EBV infection comprising administering to the patient a population of engineered T cells, the engineered T cells comprising reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, the one or more CARs comprising an extracellular ligand binding domain having specificity for CD19 or CD22, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

3. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

4. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of beta-2-microglobulin (B2M) relative to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

5. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and MHC class II transcription activator (CIITA) compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

6. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and MHC class II transcription activator (CIITA) compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

7. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

8. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

9. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type cells or a control cell, reduced expression of TCR-alpha (TRAC) and / or TCR-beta (TRB) compared to unaltered or unmodified wild-type cells or a control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, the one or more CARs comprising an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

10. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

11. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

12. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, the one or more CARs comprising an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

13. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

14. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type cells or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type cells or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

15. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising evaluating and / or diagnosing the patient for EBV infection and optionally multiple sclerosis, and administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

16. The method of any one of claims 1 to 15, wherein the one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

17. 16. The method of claim 15, wherein the one or more CARs comprises a CD8α hinge domain having the amino acid sequence of SEQ ID NO:

9.

18. 16. The method of claim 15, wherein the one or more CARs comprises a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

19. 16. The method of claim 15, wherein the one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

20. The method of any one of claims 1 to 19, wherein the one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

21. 21. The method of claim 20, wherein the one or more CARs comprises a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO:

14.

22. 21. The method of claim 20, wherein the one or more CARs comprises a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

23. The method of any one of claims 1 to 22, wherein the one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

24. The method of claim 23, wherein the one or more CARs comprises a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:

16.

25. 24. The method of claim 23, wherein the one or more CARs comprises a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO:

17.

26. 24. The method of claim 23, wherein the one or more CARs comprises a CD3ζ signaling domain having an amino acid sequence of SEQ ID NO: 18 or 115.

27. 27. The method of any one of claims 1 to 26, wherein the one or more CARs comprise an extracellular ligand binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

28. 28. The method of any one of claims 1 to 27, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

29. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB relative to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD20 CAR having a CDR sequence of SEQ ID NO:

37.

30. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV antigen CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172.

31. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO:

117.

32. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD22 CAR having a CDR sequence of SEQ ID NO:

45.

33. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB relative to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117 and a CD22 CAR having a CDR sequence of SEQ ID NO:

45.

34. 1. A method of treating multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134, or wherein the one or more CARs have an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or wherein the CARs have an scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

35. 35. The method of any one of claims 1-34, further comprising assessing said patient for EBV infection and optionally multiple sclerosis and / or diagnosing said patient with EBV infection and optionally multiple sclerosis prior to administering said population of engineered T cells to said patient.

36. 36. The method of claim 35, wherein the diagnosis comprises evaluating the patient for EBV infection.

37. 37. The method of claim 35 or 36, wherein the diagnosis comprises evaluating the patient for multiple sclerosis.

38. The method of any one of claims 1 to 37, wherein the treating is preventing multiple sclerosis.

39. The method of any one of claims 1 to 37, wherein said treating is treating multiple sclerosis.

40. The method of any one of claims 1 to 39, wherein the patient with EBV infection has been diagnosed with multiple sclerosis.

41. 41. The method of any one of claims 1 to 40, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis, progressive relapsing multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis.

42. 42. The method of any one of claims 1-41, wherein the patient achieves remission of multiple sclerosis following administration of the engineered T cells.

43. The method of any one of claims 1 to 42, wherein the patient with EBV infection is undergoing treatment for said EBV infection.

44. The method of any one of claims 1 to 43, wherein the EBV infected patient has an active EBV infection.

45. The method of any one of claims 1 to 43, wherein the EBV infected patient has an inactive EBV infection.

46. 46. ​​The method of any one of claims 1-45, wherein the patient has a reduced EBV infection following administration of the engineered T cells, and optionally the reduced EBV infection is characterized by a reduced viral load.

47. 47. The method of any one of claims 1 to 46, wherein said treating prevents conversion of EBV infection from an inactive EBV infection to an active EBV infection.

48. The method of any one of claims 1 to 47, wherein the method results in B cell depletion.

49. 49. The method of any one of claims 1-48, wherein the engineered T cells comprise one or more of 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, and an EBV antigen-specific CAR.

50. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, the one or more CARs comprising an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

51. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

52. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

53. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD20, CD22, BCMA, or EBV antigens, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

54. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

55. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

56. The method of any one of claims 50-55, wherein the one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

57. 57. The method of claim 56, wherein the one or more CARs comprises a CD8α hinge domain having the amino acid sequence of SEQ ID NO:

9.

58. 57. The method of claim 56, wherein the one or more CARs comprises a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

59. 57. The method of claim 56, wherein the one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

60. 60. The method of any one of claims 50-59, wherein the one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

61. 61. The method of claim 60, wherein the one or more CARs comprises a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO:

14.

62. 61. The method of claim 60, wherein the one or more CARs comprises a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

63. The method of any one of claims 50-62, wherein the one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

64. 64. The method of claim 63, wherein the one or more CARs comprises a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:

16.

65. 64. The method of claim 63, wherein the one or more CARs comprises a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO:

17.

66. 64. The method of claim 63, wherein the one or more CARs comprises a CD3ζ signaling domain having an amino acid sequence of SEQ ID NO: 18 or 115.

67. 67. The method of any one of claims 50-66, wherein the one or more CARs comprise an extracellular ligand binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

68. 68. The method of any one of claims 50-67, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

69. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

70. 70. The method of claim 69, further comprising evaluating said patient for and / or diagnosing said patient with said autoimmune disease.

71. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC relative to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117 and a CD22 CAR having a CDR sequence of SEQ ID NO: 45, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

72. 72. The method of any one of claims 69-71, wherein the encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 117 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 SEQ ID NO: 117 and comprises the following components: a CD8α signal peptide, an FMC63 scFv (VL-Whitlow linker-VH), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

73. 73. The method of any one of claims 69-72, wherein the encoded CD22 CAR has a corresponding amino acid sequence set forth in SEQ ID NO:45 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 SEQ ID NO:

45.

74. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV-antigen-specific CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172, and wherein the autoimmune disease is multiple sclerosis.

75. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or wherein the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

76. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

77. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

78. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

79. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

80. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

81. 1. A method of treating an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, comprising administering to the patient a population of engineered T cells, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

82. 82. The method of any one of claims 74-81, further comprising assessing and / or diagnosing said patient for said autoimmune disease prior to administering said population of engineered T cells to said patient.

83. 83. The method of any one of claims 74-82, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic scleroderma, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, and pulmonary conditions.

84. The method of any one of claims 74 to 83, wherein the patient is suspected of having or has been diagnosed as having an EBV infection.

85. 85. The method of any one of claims 74 to 84, wherein the patient is suspected of having or has been diagnosed with multiple sclerosis.

86. 86. The method of any one of claims 1-85, further comprising administering a second, third, fourth, fifth, or sixth dose of the engineered T cells to the patient.

87. 87. The method of claim 86, wherein the CAR is the same for one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells.

88. 87. The method of claim 86, wherein the CAR is different in one or more of the first, the second, the third, the fourth, the fifth, and / or the sixth dose of engineered T cells.

89. 89. The method of any one of claims 1 to 88, wherein the CAR has an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has an scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

90. 90. The method of any one of claims 1 to 89, wherein the CAR has the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, and 134.

91. 91. The method of any one of claims 1 to 90, wherein the engineered T cells comprise a CD19-specific CAR and a CD20-specific CAR.

92. 92. The method of claim 91, wherein the CD19-specific CAR has the CDR sequences of SEQ ID NO: 117 and the CD22 CAR has the CDR sequences of SEQ ID NO:

45.

93. 93. The method of claim 92, wherein the CD19-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

94. 93. The method of claim 92, wherein the CD19-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

95. 93. The method of claim 92, wherein the CD19-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

96. 96. The method of any one of claims 91-95, wherein the CD19 CAR T cells and the CD20 CAR T cells are administered simultaneously.

97. 96. The method of any one of claims 91-95, wherein the CD19 CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

98. 98. The method of claim 97, wherein the CD19 CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

99. 98. The method of claim 97, wherein the CD20 CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

100. 100. The method of any one of claims 91-99, wherein the number of cells administered as a therapeutically effective amount of the CD19 and / or CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of the CD19 CAR T cells or the CD20 CAR T cells alone.

101. 101. The method of any one of claims 91-100, wherein the number of cells administered as a therapeutically effective amount of the CD19 and / or the CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of the CD19 CAR T cells or the CD20 CAR T cells alone.

102. 102. The method of any one of claims 1 to 101, wherein the engineered T cells comprise an EBV antigen-specific CAR and a CD20-specific CAR.

103. The method of claim 102, wherein the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

104. The method of claim 102, wherein the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

105. The method of claim 102, wherein the EBV antigen-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

106. 107. The method of any one of claims 102-106, wherein the EBV-antigen CAR T cells and the CD20 CAR T cells are administered simultaneously.

107. 107. The method of any one of claims 102-106, wherein the EBV-antigen CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

108. The method of claim 107, wherein the EBV-antigen CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

109. The method of claim 107, wherein the CD20 CAR+ T cells are administered prior to administration of the EBV-antigen CAR+ T cells.

110. 110. The method of any one of claims 102 to 109, wherein the number of cells administered as a therapeutically effective amount of the EBV antigen and / or the CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of the EBV antigen CAR T cells or the CD20 CAR T cells alone.

111. 110. The method of any one of claims 102 to 109, wherein the number of cells administered as a therapeutically effective amount of the EBV antigen and / or the CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of the EBV antigen CAR T cells or the CD20 CAR T cells alone.

112. The method of any one of claims 1 to 111, wherein the engineered T cells comprise an EBV antigen-specific CAR and a CD19-specific CAR.

113. The method of claim 112, wherein the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bicistronic polynucleotide.

114. The method of claim 113, wherein the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bispecific CAR.

115. The method of claim 113, wherein the EBV antigen-specific CAR and the CD19-specific CAR are encoded by two separate polynucleotides.

116. 116. The method of any one of claims 113-115, wherein the EBV-antigen CAR T cells and the CD19 CAR T cells are administered simultaneously.

117. 116. The method of any one of claims 113-115, wherein the EBV-antigen CAR+ T cells and the CD19 CAR+ T cells are administered sequentially.

118. The method of claim 117, wherein the EBV-antigen CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

119. The method of claim 117, wherein the CD19 CAR+ T cells are administered prior to administration of the EBV-antigen CAR+ T cells.

120. 120. The method of any one of claims 113-119, wherein the number of cells administered as a therapeutically effective amount of the EBV antigen and / or the CD19 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of the EBV antigen CAR T cells or the CD19 CAR T cells alone.

121. 120. The method of any one of claims 113-119, wherein the number of cells administered as a therapeutically effective amount of the EBV antigen and / or the CD19 CAR T cells is less than the number of cells administered as a therapeutically effective amount of the EBV antigen CAR T cells or the CD19 CAR T cells alone.

122. The method of any one of claims 1 to 121, wherein the engineered T cells comprise an EBV antigen-specific CAR and a CD22-specific CAR.

123. The method of claim 122, wherein the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

124. The method of claim 122, wherein the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bispecific CAR.

125. The method of claim 122, wherein the EBV antigen-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

126. 126. The method of any one of claims 122-125, wherein the EBV-antigen CAR T cells and the CD22 CAR T cells are administered simultaneously.

127. 126. The method of any one of claims 122-125, wherein the EBV-antigen CAR+ T cells and the CD22 CAR+ T cells are administered sequentially.

128. The method of claim 127, wherein the EBV-antigen CAR+ T cells are administered prior to administration of the CD22 CAR+ T cells.

129. The method of claim 127, wherein the CD22 CAR+ T cells are administered prior to administration of the EBV-antigen CAR+ T cells.

130. 130. The method of any one of claims 122-129, wherein the number of cells administered as a therapeutically effective amount of the EBV antigen and / or the CD22 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of the EBV antigen CAR T cells or the CD22 CAR T cells alone.

131. 130. The method of any one of claims 122-129, wherein the number of cells administered as a therapeutically effective amount of the EBV antigen and / or the CD22 CAR T cells is less than the number of cells administered as a therapeutically effective amount of the EBV antigen CAR T cells or the CD22 CAR T cells alone.

132. 132. The method of any one of claims 1-131, wherein the engineered T cells are primary T cells, expanded from primary T cells or their progeny, or derived from T cells differentiated from iPSCs or their progeny.

133. 133. The method of any one of claims 1 to 132, wherein the engineered T cell is a differentiated cell derived from an induced pluripotent stem cell or a progeny thereof.

134. 134. The method of claim 133, wherein the differentiated cell is a T cell or a natural killer (NK) cell.

135. 135. The method of any one of claims 1-134, wherein the engineered T cell is a primary T cell or a progeny of a primary immune cell, optionally wherein the progeny of the primary immune cell is a T cell or a NK cell.

136. 136. The method of any one of claims 1-135, wherein the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules compared to unaltered or unmodified wild-type or control cells.

137. 137. The method of any one of claims 1-136, wherein the engineered T cells comprise reduced expression of one or more MHC HLA class II molecules compared to unaltered or unmodified wild-type or control cells.

138. 138. The method of any one of claims 1-137, wherein the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules and one or more MHC HLA class II molecules compared to an unaltered or unmodified wild-type or control cell.

139. 139. The method of any one of claims 1-138, wherein the engineered T cells comprise reduced expression of B2M and / or CIITA compared to unaltered or unmodified wild-type or control cells.

140. The method of claim 139, wherein the engineered T cells do not express B2M and / or CIITA.

141. 141. The method of any one of claims 1 to 140, wherein the engineered T cells comprise reduced expression of TRAC and / or TRB.

142. 142. The method of claim 141, wherein the engineered T cells do not express TRAC and / or TRB.

143. The method of any one of claims 1 to 142, wherein the engineered T cells comprise reduced expression of TRAC.

144. The method of claim 143, wherein the engineered T cells do not express TRAC.

145. The method of any one of claims 1 to 144, wherein the engineered T cells comprise reduced expression of TRB.

146. The method of claim 145, wherein the engineered T cells do not express TRB.

147. 147. The method of any one of claims 1-146, wherein the engineered T cells comprise reduced expression of TRAC and TRB.

148. 148. The method of any one of claims 1-147, wherein the one or more tolerogenic factors are selected from the group consisting of CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, and A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, MANF, Serpinb9, and optionally wherein the one or more tolerogenic factors comprises CD47.

149. The method of any one of claims 1 to 148, wherein the CD19-specific CAR and the one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

150. The method of any one of claims 1 to 148, wherein the CD19-specific CAR and the CD47 are encoded by a single bicistronic polynucleotide.

151. The method of any one of claims 1 to 148, wherein the CD20-specific CAR and the one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

152. The method of any one of claims 1 to 148, wherein the CD20-specific CAR and the CD47 are encoded by a single bicistronic polynucleotide.

153. 153. The method of any one of claims 1 to 152, wherein one or more of the first, second and / or third exogenous polynucleotides or the bicistronic polynucleotide are inserted into a first, second and / or third specific locus of at least one allele of the cell.

154. 154. The method of claim 153, wherein the first, second and / or third specific locus is selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

155. 155. The method of claim 154, wherein the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus.

156. 155. The method of claim 154, wherein the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

157. 157. The method of any one of claims 1 to 156, wherein the first, second and / or third exogenous polynucleotide or the bicistronic polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

158. The method of claim 157, wherein the gene therapy vector is a retrovirus or a fusosome.

159. 159. The method of any one of claims 1-158, wherein the first, second and / or third exogenous polynucleotide or the bicistronic polynucleotide is introduced into the engineered T cell using CRISPR / Cas gene editing.

160. 160. The method of claim 159, wherein the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

161. The CRISPR / Cas system is selected from the group consisting of: a. optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; b. optionally selected from the group consisting of Cas9, Csn2, and Cas4; c. optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; d. optionally, Csf1, e. Optionally, selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); f. The method or dosing regimen of claim 160, optionally comprising a Cas effector protein selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

162. 162. The method of any one of claims 159-161, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

163. 163. The method of any one of claims 159-162, wherein the first, second and / or third exogenous polynucleotide or the bicistronic polynucleotide is introduced into the engineered T cell using a lentiviral vector.

164. The method of any one of claims 1-163, wherein the engineered T cells avoid NK cell-mediated cytotoxicity upon administration to the patient.

165. The method of any one of claims 1-164, wherein the engineered T cells are protected from cytolysis by mature NK cells upon administration to the patient.

166. 166. The method of any one of claims 1-165, wherein the engineered T cells evade macrophage mediated cytotoxicity, optionally wherein the macrophage mediated cytotoxicity includes phagocytosis and / or reactive oxygen species.

167. The method of any one of claims 1-166, wherein the engineered T cells do not induce an immune response against the cells upon administration to the patient.

168. 168. The method of any one of claims 1-167, wherein said administration is selected from the group consisting of intravenous injection, intramuscular injection, intravascular injection, and implantation.

169. The method of any one of claims 1-168, wherein the engineered T cells are administered to the patient before, during, or after initiation of a different therapeutic regimen.

170. 170. The method of claim 169, wherein the different treatment regimen is selected from the group consisting of re-administration of the same or different cells, and pre-treatment, co-treatment, or post-treatment with an additional agent.

171. The method of claim 170, wherein said different cell is an autologous T cell or NK cell, or a CAR-T cell expressing a first CAR that is different from a second CAR expressed by said engineered CAR-T cell.

172. The method of any one of claims 1-171, wherein the patient has been treated with an immunodepletion therapy prior to administering the engineered T cells.

173. 173. The method of claim 172, wherein the immunodepletion therapy comprises administration of fludarabine and / or cyclophosphamide.

174. The method of any one of claims 1 to 173, wherein the patient has previously undergone antibody therapy.

175. 175. The method of claim 174, wherein the antibody therapy is rituximab.

176. The immunodepletion therapy is administered in a dose of about 1 to 50 mg / m ２ 174. The method of claim 173, comprising an IV infusion of fludarabine for about 1 to 7 days.

177. The immunodepletion therapy is administered at a dose of about 1, about 5, about 10, about 20, about 30, about 40, or about 50 mg / m ２ 177. The method of claim 176, comprising an IV infusion of fludarabine for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

178. The immunodepletion therapy is administered at a dose of about 30 mg / m ２ 178. The method of claim 176 or 177, comprising an IV infusion of fludarabine for about 4 days.

179. The immunodepletion therapy is administered at a dose of about 100 to about 1000 mg / m ２ cyclophosphamide for about 1 to 7 days.

180. The immunodepletion therapy is administered at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 mg / m ２ 180. The method of claim 179, comprising an IV infusion of cyclophosphamide for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

181. The immunodepletion therapy is administered at a dose of about 500 mg / m ２ 181. The method of claim 179 or 180, comprising an IV infusion of cyclophosphamide for about 2 days.

182. At least about 40×10 ４ 182. The method of any one of claims 1-181, wherein the engineered T cells are administered to the patient.

183. At least about 40×10 ５ 183. The method of any one of claims 1-182, wherein the engineered T cells are administered to the patient.

184. 184. The method of any one of claims 1-183, wherein the engineered T cells persist in the subject for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

185. 185. The method of any one of claims 1-184, wherein the therapeutic effect of the engineered T cells lasts for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

186. 186. The method of any one of claims 1 to 185, wherein the wild type cell or the control cell is the starting material.

187. 1. Use of a population of engineered T cells to treat EBV infection in a patient suspected of having or diagnosed with EBV infection, wherein the engineered T cells comprise reduced expression of one or more major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen (HLA) molecules compared to an unaltered or unmodified wild-type cell or a control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

188. 1. Use of a population of engineered T cells for treating EBV infection in a patient suspected of having or diagnosed with EBV infection, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to an unaltered or unmodified wild-type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19 or CD22, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

189. 1. Use of a population of engineered T cells to treat EBV infection in a patient suspected of having or diagnosed with EBV infection, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

190. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

191. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

192. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

193. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen.

194. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

195. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type cells or control cells, reduced expression of TCR-alpha (TRAC) and / or TCR-beta (TRB) compared to unaltered or unmodified wild-type cells or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

196. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

197. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

198. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

199. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild-type or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

200. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild-type cells or control cells, reduced expression of TRAC and / or TRB compared to unaltered or unmodified wild-type cells or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

201. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

202. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen.

203. The use of any one of claims 187 to 202, wherein the one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

204. The use described in claim 203, wherein the one or more CARs comprises a CD8α hinge domain having the amino acid sequence of SEQ ID NO:

9.

205. The use of claim 303, wherein the one or more CARs comprises a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

206. The use of claim 203, wherein the one or more CARs comprises an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

207. The use according to any one of claims 187 to 206, wherein the one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

208. The use described in claim 207, wherein the one or more CARs comprises a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO:

14.

209. The use of claim 207, wherein the one or more CARs comprises a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

210. The use of any one of claims 187 to 206, wherein the one or more CARs comprises a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

211. The use of claim 210, wherein the one or more CARs comprises a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:

16.

212. The use of claim 210, wherein the one or more CARs comprises a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO:

17.

213. The use of claim 210, wherein the one or more CARs comprises a CD3ζ signaling domain having an amino acid sequence of SEQ ID NO: 18 or 115.

214. The use of any one of claims 187 to 213, wherein the one or more CARs comprise an extracellular ligand binding domain comprising a scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has a scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

215. The use according to any one of claims 187 to 214, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

216. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD20 CAR having a CDR sequence of SEQ ID NO:

37.

217. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV antigen CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172.

218. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO:

117.

219. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD22 CAR having a CDR sequence of SEQ ID NO:

45.

220. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC and / or TRB compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117 and a CD22 CAR having a CDR sequence of SEQ ID NO:

45.

221. 1. Use of a population of engineered T cells to treat multiple sclerosis in a patient suspected of having or diagnosed with multiple sclerosis, wherein said engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein said one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134, or said one or more CARs have an scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or said CARs have an scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

222. 222. The use of any one of claims 187 to 221, further comprising assessing said patient for EBV infection and optionally multiple sclerosis and / or diagnosing said patient with EBV infection and optionally multiple sclerosis prior to administering said population of engineered T cells to said patient.

223. The use of claim 222, wherein the diagnosis comprises evaluating the patient for EBV infection.

224. 224. The use of claim 222 or 223, wherein the diagnosis comprises evaluating the patient for multiple sclerosis.

225. 225. The use according to any one of claims 187 to 224, wherein the treatment is preventing multiple sclerosis.

226. The use according to any one of claims 187 to 224, wherein said treating is treating multiple sclerosis.

227. The use according to any one of claims 187 to 226, wherein the patient with EBV infection has been diagnosed with multiple sclerosis.

228. The use according to any one of claims 187 to 227, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis, progressive relapsing multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis.

229. The use of any one of claims 187 to 228, wherein the patient achieves remission of multiple sclerosis following administration of the engineered T cells.

230. The use according to any one of claims 187 to 229, wherein the patient with EBV infection is undergoing treatment for said EBV infection.

231. The use according to any one of claims 187 to 230, wherein the EBV infected patient has an active EBV infection.

232. The use according to any one of claims 187 to 231, wherein the EBV infected patient has an inactive EBV infection.

233. 233. The use of any one of claims 187-232, wherein the patient has reduced EBV infection following administration of the engineered T cells, optionally wherein the reduced EBV infection is characterized by a reduced viral load.

234. The use according to any one of claims 187 to 232, wherein said treatment prevents the transformation of an EBV infection from an inactive EBV infection to an active EBV infection.

235. The use according to any one of claims 187 to 234, wherein said use results in B cell depletion.

236. 236. The use of any one of claims 187 to 235, wherein the engineered T cells comprise one or more of 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, and an EBV antigen-specific CAR.

237. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, the one or more CARs comprising an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic scleroderma, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

238. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to intact or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

239. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

240. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to intact or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

241. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M compared to an unchanged or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

242. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to intact or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, BCMA, or an EBV antigen, and wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

243. The use of any one of claims 237 to 242, wherein the one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

244. The use described in claim 243, wherein the one or more CARs comprises a CD8α hinge domain having the amino acid sequence of SEQ ID NO:

9.

245. The use of claim 243, wherein the one or more CARs comprises a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

246. The use of claim 243, wherein the one or more CARs comprises an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

247. The use according to any one of claims 237 to 246, wherein the one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

248. The use described in claim 247, wherein the one or more CARs comprises a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO:

14.

249. The use of claim 247, wherein the one or more CARs comprises a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

250. The use of any one of claims 237 to 249, wherein the one or more CARs comprises a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

251. The use of claim 250, wherein said one or more CARs comprises a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:

16.

252. The use of claim 250, wherein the one or more CARs comprises a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO:

17.

253. The use of claim 250, wherein the one or more CARs comprises a CD3ζ signaling domain having an amino acid sequence of SEQ ID NO: 18 or 115.

254. The use of any one of claims 237 to 253, wherein the one or more CARs comprise an extracellular ligand binding domain comprising a scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or the CAR has a scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

255. The use of any one of claims 237 to 254, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, or 134.

256. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC compared to an unchanged or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

257. The use of claim 256, further comprising evaluating the patient for and / or diagnosing the patient with the autoimmune disease.

258. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC compared to an unchanged or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise a CD19 CAR having a CDR sequence of SEQ ID NO: 117 and a CD22 CAR having a CDR sequence of SEQ ID NO: 45, and the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

259. 259. The use of any one of claims 256 to 258, wherein said encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 117 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 SEQ ID NO: 117 and comprises the following components: CD8α signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8α hinge domain, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain.

260. 260. The use of any one of claims 256-259, wherein the encoded CD22 CAR has a corresponding amino acid sequence as set forth in SEQ ID NO:45 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 SEQ ID NO:

45.

261. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M, CIITA, and TRAC compared to an unaltered or unmodified wild type or control cell, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an EBV-antigen-specific CAR having a CDR sequence of SEQ ID NO: 133 or 134, and / or CDR sequences derived from a VH / VL sequence of SEQ ID NO: 129-132 or 135-172, and wherein the autoimmune disease is multiple sclerosis.

262. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise an exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have an scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

263. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have the scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

264. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

265. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding a tolerogenic factor, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

266. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

267. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of B2M and CIITA compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

268. 1. Use of a population of engineered T cells to treat an autoimmune disease in a patient suspected of having or diagnosed with an autoimmune disease, wherein the engineered T cells comprise reduced expression of one or more MHC class I and / or class II HLA molecules compared to unaltered or unmodified wild type or control cells, a first exogenous polynucleotide encoding CD47, and a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs have a sequence of any one of SEQ ID NOs: 32, 34, 36, and 117, or the one or more CARs have a scFv sequence of any one of SEQ ID NOs: 19, 29, or 37.

269. 269. The use of any one of claims 237 to 268, further comprising assessing said patient for and / or diagnosing said patient with said autoimmune disease prior to administering said population of engineered T cells to said patient.

270. 270. The use of any one of claims 237 to 269, wherein the autoimmune disease is selected from the group consisting of lupus, systemic lupus erythematosus, multiple sclerosis, systemic scleroderma, vasculitis, Crohn's disease, myasthenia gravis, stiff person syndrome and pulmonary conditions.

271. The use according to any one of claims 237 to 270, wherein the patient is suspected of having or has been diagnosed as having an EBV infection.

272. The use according to any one of claims 237 to 271, wherein the patient is suspected of having or has been diagnosed with multiple sclerosis.

273. 247. The use of any one of claims 237 to 246, further comprising administering a second, third, fourth, fifth, or sixth dose of the engineered T cells to the patient.

274. 274. The use of claim 273, wherein the CAR is the same in one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells.

275. 274. The use of claim 273, wherein the CAR is different in one or more of the first, second, third, fourth, fifth, and / or sixth doses of engineered T cells.

276. 276. The use of any one of claims 187 to 275, wherein the CAR has a scFv sequence of any one of SEQ ID NOs: 19, 29, 37, 45, 54, 85, 63, 72, or 118, or wherein the CAR has a scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 129-132 or 135-172.

277. The use according to any one of claims 187 to 276, wherein the CAR has the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, 91, 92, 92, 128, 133, and 134.

278. The use of any one of claims 187 to 277, wherein the engineered T cells comprise a CD19-specific CAR and a CD20-specific CAR.

279. The use of claim 278, wherein the CD19-specific CAR has the CDR sequences of SEQ ID NO: 117 and the CD22 CAR has the CDR sequences of SEQ ID NO:

45.

280. The use described in claim 278, wherein the CD19-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

281. The use of claim 278, wherein the CD19-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

282. The use described in claim 278, wherein the CD19-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

283. The use of any one of claims 278 to 282, wherein the CD19 CAR T cells and the CD20 CAR T cells are administered simultaneously.

284. The use of any one of claims 278 to 282, wherein the CD19 CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

285. The use of claim 284, wherein the CD19 CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

286. The use of claim 284, wherein the CD20 CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

287. 287. The use according to any one of claims 278 to 286, wherein the number of cells administered as a therapeutically effective amount of said CD19 and / or said CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of said CD19 CAR T cells or said CD20 CAR T cells alone.

288. 287. The use according to any one of claims 278 to 286, wherein the number of cells administered as a therapeutically effective amount of said CD19 and / or said CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of said CD19 CAR T cells or said CD20 CAR T cells alone.

289. The use of any one of claims 187 to 288, wherein the engineered T cells comprise an EBV antigen-specific CAR and a CD20-specific CAR.

290. The use described in claim 289, wherein the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bicistronic polynucleotide.

291. The use of claim 289, wherein the EBV antigen-specific CAR and the CD20-specific CAR are encoded by a single bispecific CAR.

292. The use described in claim 289, wherein the EBV antigen-specific CAR and the CD20-specific CAR are encoded by two separate polynucleotides.

293. 293. The use of any one of claims 289 to 292, wherein the EBV-antigen CAR T cells and the CD20 CAR T cells are administered simultaneously.

294. 293. The use of any one of claims 289 to 292, wherein the EBV-antigen CAR+ T cells and the CD20 CAR+ T cells are administered sequentially.

295. The use of claim 294, wherein the EBV-antigen CAR+ T cells are administered prior to administration of the CD20 CAR+ T cells.

296. The use of claim 294, wherein the CD20 CAR+ T cells are administered prior to administration of the EBV-antigen CAR+ T cells.

297. 306. The use according to any one of claims 289 to 296, wherein the number of cells administered as a therapeutically effective amount of said EBV antigen and / or said CD20 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of said EBV antigen CAR T cells or said CD20 CAR T cells alone.

298. 30. The use according to any one of claims 289 to 296, wherein the number of cells administered as a therapeutically effective amount of said EBV antigen and / or said CD20 CAR T cells is less than the number of cells administered as a therapeutically effective amount of said EBV antigen CAR T cells or said CD20 CAR T cells alone.

299. The use according to any one of claims 187 to 298, wherein the engineered T cells comprise an EBV antigen-specific CAR and a CD19-specific CAR.

300. The use described in claim 299, wherein the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bicistronic polynucleotide.

301. The use of claim 299, wherein the EBV antigen-specific CAR and the CD19-specific CAR are encoded by a single bispecific CAR.

302. The use of claim 299, wherein the EBV antigen-specific CAR and the CD19-specific CAR are encoded by two separate polynucleotides.

303. The use of any one of claims 299 to 302, wherein the EBV-antigen CAR T cells and the CD19 CAR T cells are administered simultaneously.

304. 303. The use of any one of claims 299 to 302, wherein the EBV-antigen CAR+ T cells and the CD19 CAR+ T cells are administered sequentially.

305. The use of claim 304, wherein the EBV-antigen CAR+ T cells are administered prior to administration of the CD19 CAR+ T cells.

306. The use of claim 304, wherein the CD19 CAR+ T cells are administered prior to administration of the EBV-antigen CAR+ T cells.

307. 307. The use according to any one of claims 299 to 306, wherein the number of cells administered as a therapeutically effective amount of said EBV antigen and / or said CD19 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of said EBV antigen CAR T cells or said CD19 CAR T cells alone.

308. 307. The use according to any one of claims 299 to 306, wherein the number of cells administered as a therapeutically effective amount of said EBV antigen and / or said CD19 CAR T cells is less than the number of cells administered as a therapeutically effective amount of said EBV antigen CAR T cells or said CD19 CAR T cells alone.

309. The use of any one of claims 187 to 308, wherein the engineered T cells comprise an EBV antigen-specific CAR and a CD22-specific CAR.

310. The use described in claim 309, wherein the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

311. The use of claim 309, wherein the EBV antigen-specific CAR and the CD22-specific CAR are encoded by a single bispecific CAR.

312. The use described in claim 309, wherein the EBV antigen-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

313. The use of any one of claims 309 to 312, wherein the EBV-antigen CAR T cells and the CD22 CAR T cells are administered simultaneously.

314. 313. The use of any one of claims 309 to 312, wherein the EBV-antigen CAR+ T cells and the CD22 CAR+ T cells are administered sequentially.

315. The use of claim 314, wherein the EBV-antigen CAR+ T cells are administered prior to administration of the CD22 CAR+ T cells.

316. The use of claim 314, wherein the CD22 CAR+ T cells are administered prior to administration of the EBV-antigen CAR+ T cells.

317. 317. The use according to any one of claims 309 to 316, wherein the number of cells administered as a therapeutically effective amount of said EBV antigen and / or said CD22 CAR T cells is greater than the number of cells administered as a therapeutically effective amount of said EBV antigen CAR T cells or said CD22 CAR T cells alone.

318. 317. The use according to any one of claims 309 to 316, wherein the number of cells administered as a therapeutically effective amount of said EBV antigen and / or said CD22 CAR T cells is less than the number of cells administered as a therapeutically effective amount of said EBV antigen CAR T cells or said CD22 CAR T cells alone.

319. 319. The use of any one of claims 187 to 318, wherein the engineered T cells are primary T cells, expanded from primary T cells or their progeny, or derived from T cells differentiated from iPSCs or their progeny.

320. The use according to any one of claims 187 to 318, wherein the engineered T cell is a differentiated cell derived from an induced pluripotent stem cell or a progeny thereof.

321. The use of claim 320, wherein the differentiated cell is a T cell or a natural killer (NK) cell.

322. 322. The use of any one of claims 187 to 321, wherein the engineered T cell is a primary T cell and is a progeny of a primary immune cell, optionally wherein the progeny of the primary immune cell is a T cell or a NK cell.

323. 323. The use of any one of claims 187 to 322, wherein the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules compared to unaltered or unmodified wild-type or control cells.

324. 324. The use of any one of claims 187 to 323, wherein the engineered T cells comprise reduced expression of one or more MHC HLA class II molecules compared to unaltered or unmodified wild-type or control cells.

325. 325. The use of any one of claims 187 to 324, wherein the engineered T cells comprise reduced expression of one or more MHC HLA class I molecules and one or more MHC HLA class II molecules compared to an unaltered or unmodified wild-type or control cell.

326. The use of any one of claims 187 to 325, wherein the engineered T cells comprise reduced expression of B2M and / or CIITA compared to unaltered or unmodified wild-type or control cells.

327. The use of claim 326, wherein the engineered T cells do not express B2M and / or CIITA.

328. The use of any one of claims 187 to 327, wherein the engineered T cells comprise reduced expression of TRAC and / or TRB.

329. The use of claim 328, wherein the engineered T cells do not express TRAC and / or TRB.

330. The use of any one of claims 187 to 329, wherein the engineered T cells comprise reduced expression of TRAC.

331. The use of claim 330, wherein the engineered T cells do not express TRAC.

332. The use of any one of claims 187 to 331, wherein the engineered T cells comprise reduced expression of TRB.

333. The use of claim 332, wherein the engineered T cells do not express TRB.

334. The use of any one of claims 187 to 333, wherein the engineered T cells comprise reduced expression of TRAC and TRB.

335. 335. The use of any one of claims 187 to 334, wherein the one or more tolerogenic factors are selected from the group consisting of CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1 inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, A20 / TNFAIP3, CD39, CR1, HLA-F, IL15-RF, MANF, and Serpinb9, and optionally the one or more tolerogenic factors comprise CD47.

336. The use according to any one of claims 187 to 335, wherein the CD19-specific CAR and the one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

337. The use according to any one of claims 187 to 335, wherein said CD19-specific CAR and said CD47 are encoded by a single bicistronic polynucleotide.

338. The use according to any one of claims 187 to 335, wherein the CD20-specific CAR and the one or more tolerogenic factors are encoded by a single bicistronic polynucleotide.

339. The use according to any one of claims 187 to 335, wherein the CD20-specific CAR and the CD47 are encoded by a single bicistronic polynucleotide.

340. 340. The use according to any one of claims 187 to 339, wherein one or more of the first, second and / or third exogenous polynucleotides or the bicistronic polynucleotide are inserted into the first, second and / or third specific locus of at least one allele of the cell.

341. The use described in claim 340, wherein the first, second and / or third specific locus is selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

342. The use of claim 341, wherein the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus.

343. The use of claim 341, wherein the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

344. 344. The use according to any one of claims 187 to 343, wherein the first, second and / or third exogenous polynucleotide or the bicistronic polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

345. The use of claim 344, wherein the gene therapy vector is a retrovirus or a fusosome.

346. 346. The use of any one of claims 187 to 345, wherein the first, second and / or third exogenous polynucleotide or the bicistronic polynucleotide is introduced into the engineered T cell using CRISPR / Cas gene editing.

347. The use of claim 346, wherein the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

348. The CRISPR / Cas system is selected from the group consisting of: a. optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; b. optionally selected from the group consisting of Cas9, Csn2, and Cas4; c. optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; d. optionally, Csf1, e. Optionally, selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); f. The use or dosing regimen of claim 347, optionally comprising a Cas effector protein selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

349. The use of any one of claims 346 to 348, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

350. 350. The use of any one of claims 346 to 349, wherein the first, second and / or third exogenous polynucleotide or the bicistronic polynucleotide is introduced into the engineered T cell using a lentiviral vector.

351. The use of any one of claims 187 to 350, wherein the engineered T cells avoid NK cell mediated cytotoxicity upon administration to the patient.

352. The use of any one of claims 187 to 351, wherein the engineered T cells are protected from cytolysis by mature NK cells upon administration to the patient.

353. 353. The use of any one of claims 187 to 352, wherein the engineered T cells avoid macrophage mediated cytotoxicity, optionally wherein the macrophage mediated cytotoxicity includes phagocytosis and / or reactive oxygen species.

354. The use of any one of claims 187 to 353, wherein said engineered T cells do not induce an immune response against said cells upon administration to said patient.

355. The use according to any one of claims 187 to 354, wherein said administration is selected from the group consisting of intravenous injection, intramuscular injection, intravascular injection, and implantation.

356. The use of any one of claims 187 to 355, wherein said engineered T cells are administered to said patient before, during or after initiation of a different therapeutic regimen.

357. The use of claim 356, wherein the different treatment regimen is selected from the group consisting of re-administration of the same or different cells, and pre-treatment, co-treatment, or post-treatment with an additional agent.

358. The use of claim 357, wherein said different cell is an autologous T cell or NK cell, or a CAR-T cell expressing a first CAR that is different from the second CAR expressed by said engineered CAR-T cell.

359. The use of any one of claims 187 to 358, wherein the patient has been treated with an immunodepletion therapy prior to administering the engineered T cells.

360. The use of claim 359, wherein the immunodepletion therapy comprises administration of fludarabine and / or cyclophosphamide.

361. The use of any one of claims 187 to 360, wherein the patient has previously undergone antibody therapy.

362. The use of claim 361, wherein the antibody therapy is rituximab.

363. The immunodepletion therapy is administered in a dose of about 1 to 50 mg / m ２ The use of claim 360, comprising an IV infusion of fludarabine for about 1 to 7 days.

364. The immunodepletion therapy is administered at a dose of about 1, about 5, about 10, about 20, about 30, about 40, or about 50 mg / m ２ The use of claim 363, comprising an IV infusion of fludarabine for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

365. The immunodepletion therapy is administered at a dose of about 30 mg / m ２ 365. The use of claim 363 or 364, comprising an IV infusion of fludarabine for about 4 days.

366. The immunodepletion therapy is administered at a dose of about 100 to about 1000 mg / m ２ The use of claim 360, comprising an IV infusion of cyclophosphamide for about 1 to 7 days.

367. The immunodepletion therapy is administered at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 mg / m ２ cyclophosphamide for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days.

368. The immunodepletion therapy is administered at a dose of about 500 mg / m ２ cyclophosphamide for about 2 days.

369. At least about 40×10 ４ The use of any one of claims 187 to 368, wherein the engineered T cells are administered to the patient.

370. At least about 40×10 ５ The use of any one of claims 187 to 369, wherein the engineered T cells are administered to the patient.

371. 371. The use of any one of claims 187-370, wherein the engineered T cells persist in the subject for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

372. 372. The use of any one of claims 187-371, wherein the therapeutic effect of the engineered T cells lasts for at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer.

373. The use according to any one of claims 187 to 372, wherein said wild type cells or said control cells are the starting material.

374. 373. The method of any one of claims 1 to 186 or the use of any one of claims 187 to 373, wherein the population of engineered T cells comprises engineered T cells comprising one or more modifications that (i) reduce expression of one or more MHC class I molecules and / or one or more MHC class II molecules and / or (ii) increase expression of one or more tolerogenic factors, wherein the reduced expression of (i) and increased expression of (ii) are compared to comparable T cells not comprising said modifications.

375. An engineered T cell comprising one or more modifications that (i) reduce expression of one or more MHC class I molecules and / or one or more MHC class II molecules and / or (ii) increase expression of one or more tolerogenic factors, wherein the reduced expression of (i) and increased expression of (ii) are compared to a comparable T cell that does not contain the modification.

376. 1. A method comprising administering to a patient a population of engineered cells, the population of engineered cells comprising engineered cells that contain one or more modifications that (i) disrupt one or more MHC class I molecules and / or one or more MHC class II molecules and / or (ii) increase expression of one or more tolerogenic factors, wherein the increased expression of (ii) is compared to a comparable T cell that does not contain the modification.

377. The method of claim 376, wherein the method is a method of treating a patient suspected of having or diagnosed with an autoimmune disease.

378. 378. The method of claim 377, wherein the autoimmune disease is selected from the group consisting of Epstein-Barr virus (EBV), multiple sclerosis, lupus, systemic lupus erythematosus, multiple sclerosis, systemic scleroderma, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, or a pulmonary condition.

379. The method of any one of claims 376-378, wherein the engineered cell is a T cell or a NK cell, and optionally the T cell is a cytotoxic T cell, a helper T cell, a memory T cell, a central memory T cell, an effector memory T cell, an effector memory RAT cell, a regulatory T cell, or a tissue infiltrating lymphocyte.

380. 380. The method of any one of claims 376-379, wherein the engineered cell is a primary cell or a differentiated cell.

381. The method of any one of claims 376 to 380, wherein the one or more modifications that disrupt the one or more MHC class I molecules and / or one or more MHC class II molecules reduce expression of the one or more MHC class I molecules and / or one or more MHC class II molecules.

382. 382. The method of any one of claims 376-381, wherein the one or more tolerogenic factors are selected from the group consisting of A20 / TNFAIP3, C1 inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-F, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-L1, Serpinb9, and any combination thereof.

383. 383. The method of any one of claims 376-382, wherein said engineered cell further comprises an exogenous polynucleotide encoding one or more chimeric antigen receptors (CAR), chimeric autoantibody receptors (CAAR), or chimeric B cell autoantibody receptors (BAR).

384. The method of claim 383, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD22, CD20, BCMA, an EBV antigen, CD27, CD30, CD19 and CD20, CD19 and CD22, CD19 and CD27, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, gH / gL, EBNA1 and LMP1, EBNA1 and LMP2A, EBNA1 and LMP1 and LMP2A, LMP and BARF1 and EBNA1, CD19 and EBV antigen, CD20 and EBV antigen, or CD22 and EBV antigen.

385. The method of claim 383 or 384, wherein the one or more CAARs comprise an antigen selected from the group consisting of pancreatic beta cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and the alpha 3 chain of basement membrane collagen.

386. The method of any one of claims 383-385, wherein the one or more BARs comprises a FVIII antigen.

387. The method of claim 383 or 384, wherein the autoimmune disease is EBV and the one or more CARs comprise an extracellular ligand binding domain with specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

388. The method of claim 383 or 384, wherein the autoimmune disease is EBV and the one or more CARs comprise an extracellular ligand binding domain with specificity for CD19 or CD22, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

389. The method of claim 383 or 384, wherein the autoimmune disease is multiple sclerosis, and the one or more CARs comprise an extracellular ligand binding domain with specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

390. The method of claim 383 or 384, wherein the autoimmune disease is multiple sclerosis, and the one or more CARs comprise an extracellular ligand binding domain with specificity for a CD19, CD20, CD22, BCMA, or EBV antigen, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

391. 391. The method of any one of claims 376-390, further comprising evaluating the patient for and / or diagnosing the patient with EBV, multiple sclerosis, lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff person syndrome, or a pulmonary condition.

392. The method of any one of claims 376-391, wherein the engineered cells further comprise one or more modifications that disrupt TCR-alpha (TRAC) and / or TCR-beta (TRB).

393. The method of claim 392, wherein the one or more modifications that disrupt TCR-alpha (TRAC) and / or TCR-beta (TRB) reduce expression of TCR-alpha (TRAC) and / or TCR-beta (TRB).

394. The method of any one of claims 376-393, further comprising administering a second therapeutic agent to the patient.

395. The method of claim 394, wherein the second therapeutic agent is an anti-BLyS therapy.

396. An engineered cell comprising one or more modifications that (i) disrupt one or more MHC class I molecules and / or one or more MHC class II molecules and / or (ii) increase expression of one or more tolerogenic factors, wherein the increased expression of (ii) is compared to a comparable T cell that does not contain the modification.

397. The engineered cell of claim 396, wherein the engineered cell is a T cell or a NK cell, optionally wherein the T cell is a cytotoxic T cell, a helper T cell, a memory T cell, a central memory T cell, an effector memory T cell, an effector memory RAT cell, a regulatory T cell, or a tissue infiltrating lymphocyte.

398. The engineered cell of claim 396 or claim 397, wherein the engineered cell is a primary cell or a differentiated cell.

399. 400. The engineered cell of any one of claims 396-398, wherein the one or more modifications that disrupt the one or more MHC class I molecules and / or one or more MHC class II molecules reduce expression of the one or more MHC class I molecules and / or one or more MHC class II molecules.

400. 400. The engineered cell of any one of claims 396-399, wherein the one or more tolerogenic factors are selected from the group consisting of A20 / TNFAIP3, C1 inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-F, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-L1, Serpinb9, and any combination thereof.

401. 401. The engineered cell of any one of claims 396-400, wherein the engineered cell further comprises an exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), chimeric autoantibody receptors (CAARs), or chimeric B cell autoantibody receptors (BARs).

402. 402. The engineered cell of claim 401, wherein the one or more CARs comprise an extracellular ligand binding domain having specificity for CD19, CD22, CD20, BCMA, an EBV antigen, CD27, CD30, CD19 and CD20, CD19 and CD22, CD19 and CD27, EBNA1, EBNA3A, BRLF1, BALF4, EBNA3C, LMP1, LMP2, LMP2A, LMP2B, BZLF1, BMLF1, gp350, gH / gL, EBNA1 and LMP1, EBNA1 and LMP2A, EBNA1 and LMP1 and LMP2A, LMP and BARF1 and EBNA1, CD19 and EBV antigen, CD20 and EBV antigen, or CD22 and EBV antigen.

403. 403. The engineered cell of claim 401 or 402, wherein the one or more CAARs comprise an antigen selected from the group consisting of pancreatic beta cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, myelin oligodendritic glycoprotein, MuSK, keratinocyte adhesion protein desmoglein 3 (Dsg3), Ro-RNP complex, La antigen, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, and the alpha 3 chain of basement membrane collagen.

404. The engineered cell of any one of claims 401-403, wherein the one or more BARs comprises a FVIII antigen.

405. The engineered cell of any one of claims 396-404, wherein the engineered cell further comprises one or more modifications that disrupt TCR-alpha (TRAC) and / or TCR-beta (TRB).

406. The engineered cell of claim 405, wherein the one or more modifications that disrupt TCR-alpha (TRAC) and / or TCR-beta (TRB) reduce expression of TCR-alpha (TRAC) and / or TCR-beta (TRB).

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