Methods and compositions for treating autoimmune diseases

Abstract

The invention relates generally to methods of treating autoimmune disease with immune cell therapy. The invention also relates generally to methods of treating autoimmune disease which do not include administering to the subject a preconditioning regimen prior to the immune cell therapy.

Description

METHODS AND COMPOSITIONS FOR TREATING AUTOIMMUNE DISEASESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 567,674, filed March 20, 2024, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on March 17, 2025, is named CBB-204WO_SL.xml and is 141 kilobytes in size.FIELD OF THE INVENTION

[0003] The invention relates generally to methods of using immune cell therapy to treat autoimmune diseases in a subject, including methods that do not include administering to the subject (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, and / or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine.BACKGROUND

[0004] According to the National Institute of Health, autoimmune diseases affect more than 23.5 million people in the United States. Autoimmune diseases are a group of diseases which result from body’s immune system attacking healthy cells or tissues. In the case of autoimmune diseases where B cells have a role in initiating or maintaining the disease, certain populations of a patient’s B cells differentiate into antibody secreting cells that produce antibodies directed against normal tissues and cells. While these autoantibodies are the effectors of such diseases, the underlying cause is defective B cells that mistakenly differentiate into cells that secrete these pathogenic antibodies.

[0005] Current treatment options for autoimmune mediated diseases involve generalized immune suppression, achieved through corticosteroids, immunosuppressive medications, and biologies. Most commonly, corticosteroids are used on both a chronic and acute basis to control disease and act via a variety of mechanisms to control or downregulate multiple inflammatorypathways. In many cases, systemic immunosuppressive medications such as mycophenolate, azathioprine and methotrexate, are added in an effort to minimize symptoms and manage the expected recurrences in patients. Most therapies for treating autoimmune diseases are not curative and require chronic administration, often for the lifetime of the patient. Furthermore, there is the risk of serious and life-threatening infection for patients receiving chronic immunosuppressive therapy. As such, there is a significant unmet medical need for these patients.

[0006] Over the course of the last decade, chimeric antigen receptor (CAR) T cell therapy has become an established treatment for a variety of cancers. The method has seen particular success in haematological malignancies such as acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In general, CAR T cell therapy for the treatment of cancer involves isolating T cells from human blood, genetically engineering the cells to express a CAR against a desired target antigen, administering a lymphodepleting chemotherapeutic preconditioning regimen to the patient, and subsequently administering the engineered CAR T cells to the patient, thereby allowing the CAR T cells to target and destroy cancerous cells expressing this antigen. Lymphodepleting preconditioning has been considered to be an integral part of CAR T cell therapy, and it has been demonstrated that such a preconditioning regimen enhances the survival, persistence, and efficacy of the administered CAR T cells, for example, by increasing levels of homeostatic cytokines such as IL-2, IL-7, and IL- 15, and by eliminating endogenous immune cells that compete for such cytokines, thereby creating a more favorable environment for the infused cells to proliferate and persist (Nissani et al. (2021) J. IMMUNOTHER. CANCER 9: e001743). Additional strategies for enhancing CAR T cell persistence in cancer therapies include the use of IL-2, IL-7, IL- 15, or related variants or signaling proteins. These strategies include, for example, administering IL- 15 or IL-2 following CAR T infusion, engineering CAR T cells to ectopically express IL- 15 or IL-7, tethering IL- 15 to the membrane of administered CAR T cells, and engineering the CAR T cells to express recombinant cytokine receptor signaling proteins (e.g., recombinant IL-15R or recombinant IL-2R) (Bell et al. (2021) FRONT. IMMUNOL. 12: 684642).

[0007] More recently, CAR T cell therapy has also been investigated as treatment for autoimmune diseases, including refractory systemic lupus erythematosus (SLE), a B-cell- mediated autoimmune disease. Researchers reported that administration of a CAR T cell therapyinduced clinical remission in 5 out of 5 patients with moderate to severe, refractory SLE (Mackensen et al. (2022) NAT. MED. 28: 2124-2132). In this study, patients received a lymphodepleting preconditioning regimen prior to the CAR T cell therapy, comprising doses of both fludarabine and cyclophosphamide (Mackensen, supra). Similar findings were reported in antisynthetase syndrome (myositis) (Muller et al. (2023) LANCET 401 : 815-818). The requirement for the preconditioning regimen in achieving a successful therapeutic outcome was not investigated in either study. Furthermore, recently disclosed clinical trials investigating the treatment of autoimmune diseases with genetically modified, autologous CD19-CAR T cells also utilize a lymphodepleting preconditioning regimen (see, e.g., ClinicalTrials.gov ID Nos. NCT06121297, NCT06154252, NCT06138132, NCT06152172, NCT05938725, and NCT06193889).

[0008] Although lymphodepleting preconditioning is considered in the art to be a prerequisite for successfully treating autoimmune diseases with engineered T cell therapies, the role and importance of lymphodepleting preconditioning in enabling the successful treatment of autoimmune diseases with T cell therapy is not understood. Despite of the advancements made to date, there remains a need for new, useful and tolerable treatments for treating autoimmune diseases.SUMMARY OF THE INVENTION

[0009] Although preconditioning regimens have been used in engineered immune-cell therapies for treating cancer, given the potential mortality of subjects with cancer, aggressive preconditioning regimens have been considered to pose an acceptable risk to the patients.However, autoimmune diseases generally do not pose the same imminent life-threatening risk as cancer. As a result, preconditioning treatments suitable for treating cancer may not be as appropriate for treating patients with autoimmune diseases, and thus the associated risk of such a preconditioning regime may not be acceptable to the patients and their physicians. Furthermore, other strategies for enhancing persistence of CAR immune cells in the context of cancer treatment, such as administering exogenous cytokines or modifying cells to express cytokines, may not be suitable for treating autoimmune diseases, as they could potentially worsen a subject’s autoimmune disease through the activation or potentiation of autoreactive T cells.

[0010] It has now been discovered that autoimmune diseases can be effectively treated with CAR immune cell therapies, even without administering a lymphodepleting preconditioning regimen or cytokines. The methods of treatment described herein, which include administering a genetically modified CAR immune cell to a subject without administering a lymphodepleting preconditioning regimen or cytokines, are surprisingly found to enable persistence of the CAR immune cells in the subject at sufficient levels to enable successful treatment of autoimmune diseases, while eliminating the toxicity and risks normally associated with standard preconditioning regimens and cytokine treatments.

[0011] Accordingly, in one aspect, the present disclosure provides a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain comprising a binding domain that binds a B-cell surface protein, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine, wherein the treatment establishes a population of genetically modified immune cells in the subject, and (a) the abundance of the DNA encoding the CAR reaches at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 1,000 copies per microgram of PBMC genomic DNA for at least 5 days; and / or (b) the population of genetically modified immune cells is established in the subject at a concentration of at least 100 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 5 days.

[0012] In certain embodiments of any of the foregoing methods, the DNA encoding the CAR remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 days, at least 28, or at least 29 days. In certain embodiments, the abundance of the DNA encoding the CAR reaches at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 copies per microgram of genomic DNA in PBMCs harvested from the subject. For example, in certain embodiments, the abundance of theDNA encoding the CAR reaches at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 3,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days. In certain embodiments, the abundance of the DNA encoding the CAR reaches at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 5,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days. In further embodiments, the abundance of the DNA encoding the CAR reaches at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 10,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

[0013] In certain embodiments of any of the foregoing methods, the genetically modified immune cell is administered to the subject on Day 0, and the DNA encoding the CAR remains at an abundance of at least 1,000 (e.g., at least 3,000, at least 5,000, or at least 10,000) copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, from Day 5 to Day 21, or from Day 5 to Day 29.

[0014] In certain embodiments of any of the foregoing methods, the abundance of the DNA encoding the CAR in PBMCs harvested from the subject is measured by quantitative PCR (qPCR).

[0015] In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 6 days, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

[0016] In certain embodiments, the population of genetically modified immune cells in the subject is established at a concentration of at least 200, at least 300, at least 400, or at least 500 cells per microliter of blood. For example, in certain embodiments, the population of geneticallymodified immune cells in the subject is established at a concentration of at least 200 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 200 cells per microliter of blood for at least 6 days, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days. In further embodiments, the population of genetically modified immune cells is established in the subject at a concentration of at least 300 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood for at least 6 days, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

[0017] In certain embodiments of any of the foregoing methods, the genetically modified immune cell is administered to the subject on Day 0, and the population of genetically modified immune cells persists in the subject at a concentration of at least 100 (e.g., at least 200 or at least 300) cells per microliter of blood from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

[0018] In certain embodiments of any of the foregoing methods, the concentration of genetically modified immune cells in the blood of the subject is measured by flow cytometry.

[0019] In certain embodiments of any of the foregoing methods, the subject has not received a dose of cyclophosphamide and / or fludarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months prior to administration of the genetically modified immune cell.

[0020] In certain embodiments of any of the foregoing methods, the method does not include administering a chemotherapeutic agent to the subject before or during administration of the genetically modified immune cell. In certain embodiments, the method does not include treating the subject with radiation therapy before or during administration of the genetically modified immune cell. In certain embodiments, the method does not include exposing the subject to total lymphatic irradiation before or during administration of the genetically modified immune cell.

[0021] In certain embodiments of any of the foregoing methods, the genetically modified immune cell is not engineered to ectopically express a cytokine-pathway signaling protein. Incertain embodiments, the genetically modified immune cell is not engineered to ectopically express a cytokine receptor. In certain embodiments, the genetically modified immune cell is not engineered to ectopically express a protein (e.g., a recombinant protein) other than the CAR.

[0022] In certain embodiments of any of the foregoing methods, the genetically modified immune cell is not transfected with an mRNA prior to being administered to the subject.

[0023] In certain embodiments of any of the foregoing methods, the method does not include administering a recombinant cytokine to the subject before or during administration of the genetically modified immune cell.

[0024] In certain embodiments of any of the foregoing methods, the B-cell surface protein is CD 19.

[0025] In certain embodiments of any of the foregoing methods, the extracellular binding domain of the CAR comprises an antigen-binding site that specifically binds the B-cell surface protein, wherein the antigen-binding site comprises a heavy chain variable domain (VH) comprising complementarity determining regions CDRm, CDRm, and CDRHS and a light chain variable domain (VL) comprising complementarity determining regions CDRLI, CDRL2, and CDRLS. The antigen binding site can be, e.g., humanized or fully human. In certain embodiments, the CDRm, CDRm, and CDRm, CDRLI, CDRL2, and CDRL3 comprise the amino sequences of (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively; (11) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively; or (111) SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, GAS, and SEQ ID NO: 47, respectively, and / or the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of (i) SEQ ID NOs: 4 and 8, respectively; (ii) SEQ ID NOs: 13 and 17, respectively; or (iii) SEQ ID NOs: 44 and 48, respectively. In certain embodiments, the VH and the VL comprise the amino acid sequences of: (i) SEQ ID NOs: 4 and 8, respectively; (ii) SEQ ID NOs: 13 and 17, respectively; or SEQ ID NOs: 44 and 48, respectively.

[0026] In certain embodiments of the foregoing methods, the antigen-binding site of the CAR is present in an scFv. The scFv can, for example, comprise an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51. In certain embodiments, the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 9, 18,and 51.

[0027] In certain embodiments of the foregoing methods, the B-cell surface protein comprises a MuSK autoantibody. For example, in certain embodiments, the extracellular binding domain of the CAR comprises a MuSK autoantigen, optionally a full-length MuSK autoantigen. The MuSK autoantigen can comprise, e.g., an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55. In certain embodiments, the MuSK autoantigen comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0028] In certain embodiments of any of the foregoing methods, the extracellular binding domain of the CAR does not comprise a Dsg autoantigen and / or a Dsg3 autoantigen.

[0029] In a related aspect, the present disclosure provides a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, the CAR comprising an extracellular domain comprising an antigen-binding site that specifically binds a B-cell surface protein, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine. In certain embodiments, the treatment establishes a population of genetically modified immune cells in the subject, optionally wherein the abundance of the DNA encoding the CAR reaches at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 1,000 copies per microgram of PBMC genomic DNA for at least 5 days. For example, in certain embodiments, the DNA encoding the CAR remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 6 days, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days. In certain embodiments, the abundance of the DNA encoding the CAR reaches at least 2,000, at least 3,000, at least 4,000, at least 5,000, or at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject. For example, in certain embodiments, the abundance of the DNA encoding the CAR reaches at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 3,000 copies per microgram of PBMC genomic DNA for atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days. In certain embodiments, the abundance of the DNA encoding the CAR reaches at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 5,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 days, at least 28, or at least 29 days. In further embodiments, the DNA encoding the CAR reaches at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 10,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

[0030] In certain embodiments of the foregoing methods, the genetically modified immune cell is administered to the subject on Day 0, and the DNA encoding the CAR remains at an abundance of at least 1,000 (e.g., at least 3,000, at least 5,000, or at least 10,000) copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

[0031] In certain embodiments of the foregoing methods, the abundance of the DNA encoding the CAR in PBMCs harvested from the subject is measured by quantitative PCR (qPCR).

[0032] In certain embodiments of the foregoing methods, the population of genetically modified immune cells is established in the subject at a concentration of at least 100, at least 200, at least 300, at least 400, at least 500, or at least 1,000 cells per microliter of blood.

[0033] In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 5 days. For example, in certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 6 days, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days. In certain embodiments, the population of genetically modified immune cells in the subject is established ata concentration of at least 200 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 200 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days. In further embodiments, the population of genetically modified immune cells in the subject is established at a concentration of at least 300 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

[0034] In certain embodiments of the foregoing methods, the genetically modified immune cell is administered to the subject on Day 0, and the population of genetically modified immune cells persists in the subject at a concentration of at least 100 (e.g., at least 200 or at least 300) cells per microliter of blood from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

[0035] In certain embodiments of the foregoing methods, the concentration of genetically modified immune cells in the blood of the subject is measured by flow cytometry.

[0036] In certain embodiments of the foregoing methods, the subject has not received a preconditioning regimen that includes cyclophosphamide and / or fludarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months prior to administration of the genetically modified immune cell.

[0037] In certain embodiments of the foregoing methods, the method does not include administering a chemotherapeutic agent to the subject before or during administration of the genetically modified immune cell. In certain embodiments, the method does not include treating the subject with radiation therapy before or during administration of the genetically modified immune cell. In certain embodiments, the method does not include exposing the subject to total lymphatic irradiation before or during administration of the genetically modified immune cell.

[0038] In certain embodiments of the foregoing methods, the genetically modified immune cell is not engineered to ectopically express a cytokine-pathway signaling protein. In certain embodiments, the genetically modified immune cell is not engineered to ectopically express acytokine receptor. In certain embodiments, the genetically modified immune cell is not engineered to ectopically express a protein (e.g., a recombinant protein) other than the CAR.

[0039] In certain embodiments of the foregoing methods, the genetically modified immune cell is not transfected with an mRNA prior to being administered to the subject.

[0040] In certain embodiments of the foregoing methods, the method does not include administering a recombinant cytokine to the subject before or during administration of the genetically modified immune cell.

[0041] In certain embodiments of the foregoing methods, the B-cell surface protein is CD19.

[0042] In certain embodiments of the foregoing methods, the antigen-binding site of the CAR comprises a heavy chain variable domain (VH) comprising complementarity determining regions CDRHI, CDRH2, and CDRJB and a light chain variable domain (VL) comprising complementarity determining regions CDRLI, CDRL2, and CDRLS. The antigen binding site can be, e.g., humanized or fully human. In certain embodiments, the CDRHI, CDRH2, and CDRHS, CDRLI, CDRL2, and CDRL3 comprise the amino sequences of (i) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively; (n) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively; or (hi) SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, GAS, and SEQ ID NO: 47, respectively. In certain embodiments, the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of (i) SEQ ID NOs: 4 and 8, respectively; (ii) SEQ ID NOs: 13 and 17, respectively; or (iii) SEQ ID NOs: 44 and 48, respectively. In certain embodiments, the VH and the VL comprise the amino acid sequences of: (i) SEQ ID NOs: 4 and 8, respectively; (ii) SEQ ID NOs: 13 and 17, respectively; or SEQ ID NOs: 44 and 48, respectively.

[0043] In certain embodiments of the foregoing methods, the antigen-binding site of the CAR is present in an scFv. The scFv can, for example, comprise an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51. In certain embodiments, the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51.

[0044] In certain embodiments of any of the foregoing methods, the CAR may furthercomprise a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain. The transmembrane domain can comprise, for example, a CD8 alpha chain transmembrane domain, e.g., wherein the CD8 alpha chain transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19. The costimulatory domain can comprise, for example, a 4-1BB intracellular domain, e.g., wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 20. The intracellular signaling domain can comprise, for example, a CD3 zeta signaling domain, e.g., wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 21. The CAR can also further comprise a hinge domain or linker, which may be interposed between the extracellular binding domain and the transmembrane domain. The hinge domain can be, for example, a CD8 alpha chain hinge, e.g., wherein the CD8 alpha chain hinge comprises the amino acid sequence of SEQ ID NO: 22. In certain embodiments of the foregoing methods, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 23 and 27.

[0045] In certain embodiments of any of the foregoing methods, the CAR may further comprise a killer immunoglobulin-like receptor (KIR) transmembrane domain and a KIR cytoplasmic domain.

[0046] In certain embodiments of any of the foregoing methods, the genetically modified immune cell is a T cell or an NK cell, e.g., a T cell, such as a T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a memory T cell, an alpha beta T cell, and a gamma delta T cell. In certain embodiments, the T cell is a cytotoxic T cell.

[0047] In certain embodiments of any of the foregoing methods, the genetically modified immune cell is autologous to the subject.

[0048] It is contemplated that the genetically modified immune cell can be administered to the subject in a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier or excipient.

[0049] Each of the foregoing method can comprise administering the genetically modified immune cell to the subject at a dose from 1 x 105cells / kg to 1 x 108cells / kg, e.g., at a dose from 1 x 106cells / kg to 1 x 107cells / kg.

[0050] In certain embodiments of any of the foregoing methods, the DNA encoding the CARis introduced into the genetically modified immune cell by a viral vector, such as a lentiviral vector or an adeno-associated viral vector.

[0051] In certain embodiments of any of the foregoing methods, the autoimmune disease is a B-cell-mediated autoimmune disease. For example, the autoimmune disease can be selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myasthenia gravis (MG), myositis, systemic sclerosis, multiple sclerosis, membranous nephropathy, and chronic immune demyelinating polyneuropathy. In certain embodiments, the autoimmune disease is selected from the group consisting of lupus nephritis, SLE with anti- dsDNA antibodies, mucosal PV, mucocutaneous PV, MuSK-associated MG, AChR MG, antisynthetase syndrome, dermatomyositis, and immune mediated necrotizing myopathy.

[0052] These and other aspects and features of the present disclosure are described in the following detailed description and claims.DESCRIPTION OF THE DRAWINGS

[0053] The invention can be more completely understood with reference to the following drawings.

[0054] FIGURE 1A and FIGURE IB depict schematic diagrams of exemplary anti-CD19- CAR constructs. FIGURE 1C depicts a schematic diagram of an exemplary MuSK-CAR construct.

[0055] FIGURES 2A-2D summarize persistence of B-cell-targeted CAR T cells in vivo in patients having an autoimmune disease. Patients received either 5 x 108MuSK-CAR T cells (“Al-#”) or 2.5 x 109MuSK-CAR T cells (“A2-#”) at Day 0 without any lymphodepleting preconditioning. Post-infusion MuSK-CAR T cell levels were measured by qPCR for vector sequences in genomic DNA extracted from peripheral whole blood samples, and by flow cytometry to quantify CAR-expressing T cells. By way of comparison, historical CAR-T persistence data with a Dsg-CAR, following administration of a total dose of 5 x 108(“A3-#”) or 2.5 x 109(“A4-#”) T cells transduced with a Dsg-CAR construct, indicates that the Dsg-CAR-T cells do not effectively persist in vivo (“Dsg-CAR”). FIGURE 2A is a line graph summarizing persistence of MuSK-CAR T cells in vivo in six MuSK-CAR patients, reported as vector copies per pg DNA. Shading represents levels of persistence seen when using anti-CD19-CAR T cellsto treat heme cancers with preconditioning (mean dose 3 x 108CAR-T cells). FIGURE 2B is a bar graph depicting the area under the curve (AUC) for the data shown in FIGURE 2A through Day 29. FIGURE 2C is a line graph summarizing persistence of MuSK-CAR T cells in vivo in six MuSK-CAR patients, reported as MuSK-CAR T (“MuSK-CAART”) cells per pL of blood. FIGURE 2D is a bar graph depicting the AUC for the data shown in FIGURE 2C through Day 29.

[0056] FIGURE 3 depicts a series of flow cytometry plots summarizing persistence of B- cell-targeted CAR T cells in vivo following administration to patients with an autoimmune disease. The left column summarizes the percentage of MuSK-CAR T cells in the manufactured cell therapy product (“MP”) pre-infusion. Patients were infused with the MP at Day 0, and MuSK-CAR T cell levels post-infusion were measured by flow cytometry at the indicated days. The first and second row summarize data from two patients (“Al-1” and “Al-2,” respectively). Patients were not administered a preconditioning regimen. X-axis indicates CD3 expression, and Y-axis indicates CAR expression.

[0057] FIGURE 4 depicts a line graph summarizing serum IFNy levels following administration of MuSK-CAR T cells to six patients (“Al-#” and “A2-#”) with an autoimmune disease. Patients were not administered a lymphodepleting preconditioning regimen.

[0058] FIGURE 5 depicts a line graph summarizing MuSK autoantibody levels in myasthenia gravis patients before and after administration of MuSK-CAR T cells. Patients were administered either 5 x 108(“Al-#’) or 2.5 x 109(“A2-#”) CAR T cells, and patients were not administered a lymphodepleting preconditioning regimen. Results are depicted as concentration of MuSK autoantibodies.

[0059] FIGURES 6A-6D depict line graphs summarizing myasthenia gravis clinical scores in six patients before and after administration of MuSK-CAR T cells. Patients were administered either 5 x 108(“Al-#’) or 2.5 x 109(“A2-#”) CAR T cells, and patients were not administered a lymphodepleting preconditioning regimen. The following four clinical scores were measured: Myasthenia Gravis Composite scale (MGC, FIGURE 6A), the Myasthenia Gravis Activities of Daily Living Profile (MG-ADL, FIGURE 6B), improved 15-item myasthenia gravis quality of life scale (MG-QOL 15r, FIGURE 6C), and quantitative Myasthenia Gravis scoring system (QMG, FIGURE 6D)DETAILED DESCRIPTION

[0060] Although preconditioning regimens have been used in engineered immune-cell therapies for treating cancer, given the potential mortality of subjects with cancer, aggressive preconditioning regimens have been considered to pose an acceptable risk to the patients. However, autoimmune diseases generally do not pose the same imminent life-threatening risk as cancer. As a result, preconditioning treatments suitable for treating cancer may not be as appropriate for treating patients with autoimmune diseases, and thus the associated risk of such a preconditioning regime may not be acceptable to the patients and their physicians. Furthermore, other strategies for enhancing persistence of CAR immune cells in the context of cancer treatment, such as administering exogenous cytokines or modifying cells to express cytokines, may not be suitable for treating autoimmune diseases, as they could potentially worsen a subject’s autoimmune disease through the activation or potentiation of autoreactive T cells.

[0061] It has now been discovered that autoimmune diseases can be effectively treated with CAR immune cell therapies, even without administering a lymphodepleting preconditioning regimen or cytokines to the subject. The methods of treatment described herein, which include administering a genetically modified CAR immune cell to a subject without administering a lymphodepleting preconditioning regimen or cytokines, are surprisingly found to enable persistence of the CAR immune cells in the subject at sufficient levels to enable successful treatment of autoimmune diseases, while eliminating the toxicity and risks normally associated with standard preconditioning regimens and cytokine treatments.I. Definitions

[0062] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0063] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0064] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0065] As used herein, the term “antigen-binding site” refers to an antigen-binding fragment of an immunoglobulin or a derivative or variant thereof that participates in antigen binding. For example, in human antibodies, an antigen-binding site is formed by the N-terminal variable domains of the heavy chain and light chain, which are also called “heavy chain variable domain (VH)” and “light chain variable domain (VL),” respectively. In each of the variable domains, three highly divergent stretches called “hypervariable regions” are interposed between more conserved flanking stretches known as “framework regions” (FRs). The three hypervariable regions of a VH and the three hypervariable regions of a VL are disposed relative to each other in three-dimensional space to form an antigen-binding surface complementary to the three- dimensional surface of a bound antigen. The hypervariable regions are also referred to as “complementarity-determining regions” or “CDRs.” The boundaries of the FRs and CDRs can be defined using any appropriate convention known in the art, including, for example, by the IMGT convention (see, Lefranc, (1999) The Immunologist, 7, 132-136), by Kabat convention (see, Kabat, E.A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), or by the Chothia convention (see, Chothia, C. etal. (1987) J. MOL. BIOL. 196:901-917). The three CDRs, referred to as CDRi, CDR2, and CDR3, contribute to the antibody binding specificity.

[0066] Examples of antigen-binding fragments of an immunoglobulin include, for example, Fab, Fab’, and F(ab’)2 fragments. Examples of variants of antigen-binding fragments of immunoglobulins include, for example, single chain antibodies or scFvs. Certain animals have different forms of antibodies. For example, camelids have antibodies comprising VHH fragments and cartilaginous fish have antibodies called “new antigen receptor immunoglobulins” or “IgNARs” comprising VNAR fragments, where such fragments, which are single, monomeric antibody variable domains that are able to bind selectively to a specific antigen independently of another variable domain, are called “single domain antibody,” “sdAb,” or “nanobody.” An antigen-binding site can comprise either a pair of VH and VL or an sdAb. An antigen-binding site disclosed herein can be recombinant, chimeric, deimmunized, humanized, and / or affinity matured (see, e.g., U.S. Pat. No. 4,816,567; Morrison etal. (1984) PROC. NATL. ACAD. SCI. U.S.A., 81 : 6851-55; Morrison etal. (1985) PROC. NATL. ACAD. SCI. U.S.A., 81:6851; Takeda et al. (1985) NATURE, 314: 452).

[0067] The term “autoantigen,” as used herein, refers to an endogenous antigen that stimulatesproduction of an autoimmune response, such as production of autoantibodies. Autoantigen also includes a self-antigen or antigen from a normal tissue that is the target of a cell-mediated or an antibody-mediated immune response that may result in the development of an autoimmune disease. The term “autoantigen” is understood to refer to both a full-length autoantigen as well as autoantibody-binding fragments thereof, unless otherwise specifically stated or understood from the context. Examples of autoantigens include, but are not limited to, the extracellular portion of MuSK, and autoantibody-binding fragments thereof.

[0068] The terms “chimeric antigen receptor” or “CAR,” as used herein, refer to a recombinant receptor that is expressed by a cell, e.g., an immune cell or any other effector cell type, e.g., an effector cell type capable of cell-mediated cytotoxicity, such as a T cell. The CAR includes an extracellular binding domain that specifically binds a protein on the surface of a target cell, e.g., a cell surface protein (e.g., B-cell surface protein) such as CD 19 or BCMA, or an autoantibody or B-cell receptor (BCR). The binding domain can comprise, for example, an antigen-binding site from an antibody (e.g. an anti-CD19 or anti -BCMA antibody), or an autoantigen (e.g., a MuSK autoantigen or BCR-binding fragment thereof). The CAR may also optionally include (i) a transmembrane domain, a signaling domain, and / or an intracellular costimulatory domain; or (ii) a killer immunoglobulin-like receptor (KIR) transmembrane domain and a KIR cytoplasmic domain. A CAR that comprises an extracellular binding domain comprising an autoantigen can also be referred to herein as a “chimeric autoantigen receptor” or a “CAAR.”

[0069] The term “cross-compete,” as used herein in the context of a subject antibody and a reference antibody, indicates that the subject antibody competes for binding to an antigen (e.g., CD19) with the reference antibody and vice versa. A subject antibody cross-competes with a reference antibody if competition is observed whether the reference antibody is used as the first antibody and the subject antibody is used as the second antibody, or the subject antibody is used as the first antibody and the reference antibody is used as the second antibody in this assay. A skilled artisan can select the concentrations of the antibodies used in the competition assays based on the affinities of the antibodies for the antigen and the valency of the antibodies. In an exemplary assay, a first anti-CD19 antibody is immobilized on a solid surface, CD 19 is bound to the first antibody, and binding of a second anti-CD19 antibody is assessed. If the second antibody does not generate a significant binding signal, the second antibody competes with thefirst antibody for binding CD 19. Exemplary assays are described in Cox et al., “Immunoassay Methods,” in ASSAY GUIDANCE MANUAL [INTERNET], Updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al. (2001) CYTOMETRY, 44: 30-37; and Finco etal. (2011) J. PELARM. BlOMED. ANAL., 54: 351-358. A CD 19 binding agent comprising an antigen-binding site, such as a fragment of, or scFv derived from, an anti-CD19 antibody, can also be assessed in this assay.

[0070] Doses described herein can be presented as a “weight based dose” or as a “body surface area (BSA) based dose.” A weight based dose is a dose that is administered to a patient that is calculated based on the weight of the patient, e.g., mg / kg. A BSA based dose is a dose that is administered to a patient that is calculated based on the surface area of the patient, e.g., mg / m2. The two forms of dose measurement can be converted for human dosing by multiplying the weight based dose by 37 or dividing the BSA based dose by 37. For example, a dose of 60 mg / kg to be administered to a human subject is equivalent to a 2220 mg / m2dose of the same drug to be administered to the same subject.

[0071] As used herein, the term “effective amount” refers to the amount of a compound or agent (e.g., a compound or agent of the present disclosure) sufficient to effect beneficial or desired results. For example, “effective amount” can refer to the amount of an active agent (e.g., a CAR immune cell) sufficient to effect beneficial or desired results. For example, an effective amount of an agent may be an amount sufficient to achieve one or more of the following: (1) treat an autoimmune disease, e.g, SLE, pemphigus vulgaris, myasthenia gravis, or myositis; (2) reduce or eliminate circulating B cells in a subject; (3) decrease autoantibody levels in a subject; (4) reduce or eliminate endogenous lymphocytes in a subject; (5) reduce proteinuria in a subject; or (6) increase the amount or activity of one or more complement factors in a subject. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0072] As used herein, percent “identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, percent “identity” between a nucleic acid sequence and a reference sequence is defined as thepercentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0073] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0074] As used herein, the term “isolated” when used in conjunction with a particular article (e.g., polypeptide, nucleic acid, or cell) is understood to mean: (1) that the article has been separated or purified from other components (e.g., other proteins, peptides, nucleic acids, cells, or cellular materials) and / or chemicals (e.g., reagents used in manufacture); (2) that the article may be separated or purified from the environment in which it may exist in nature, for example, a tissue or fluid sample; or (3) that the article does not occur in nature. For example, a molecule that is removed from a cell that produces it, is “isolated.” A chemically synthesized molecule is “isolated.” As used herein, the term “isolated” can also refer to a molecule that is substantially free of other molecules of the same species. For example, a protein may be “isolated” from other proteins having different amino acid sequences. The purity or homogeneity of a desired article can be assayed using techniques well known in the art, including gel electrophoresis, high performance liquid chromatography, or mass spectrometry. Any of the polynucleotides, polypeptides, vectors, compounds, or cells described herein may be isolated.

[0075] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensuratewith a reasonable benefit / risk ratio.

[0076] The term “pharmaceutically acceptable carrier” as used herein refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23 d ed. 2020).

[0077] The term “purified,” as used herein, means that an entity or substance is separated from one or more other entities or substances with which it was previously found before being purified. An entity or substance may be partially purified, substantially purified, or pure. A substance or entity such as a nucleic acid or polypeptide is considered pure when it is removed from substantially all other compounds or entities other than a solvent and any ions contained in the solvent, i.e., it constitutes at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of the dry weight of the composition. A partially or substantially purified compound or entity such as a nucleic acid or polypeptide may be removed from at least 50%, at least 60%, at least 70%, or at least 80% by weight of the material with which it is naturally found, e.g, cellular material such as cellular proteins and / or nucleic acids. In certain embodiments, the purified nucleic acid or polypeptide constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even more, by dry weight, of the total nucleic acid or polypeptide, respectively, in a composition. Methods for assessing purity are known in the art and include chromatographic methods, immunological methods, electrophoretic methods, etc. Any of the polynucleotides, polypeptides, or cells described herein may be purified.

[0078] As used herein, the terms “subject” and “patient” refer to an organism to be treated by any of the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.

[0079] As used herein, “treat,” “treating,” and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans. As used herein, “prevent,” “preventing,” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.II. Methods

[0080] Disclosed herein, in one aspect, is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell (e.g., T cell) comprising a DNA encoding a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain comprising a binding domain that specifically binds a B-cell surface protein, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine. Also disclosed herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell (e.g., T cell) comprising a DNA encoding a CAR, the CAR comprising an extracellular domain comprising an antigen-binding site that specifically binds a B-cell surface protein, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine. Also disclosed herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell (e.g., T cell) comprising a DNA encoding a CAR, the CAR comprising an extracellular domain comprising an antigen-binding site that specifically binds a B-cell surface protein, wherein the method does not include administering to the subject a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, and wherein the genetically modified immune cell is autologous to the subject. In certain embodiments of the therapeuticmethods disclosed herein, the treatment establishes a population of genetically modified immune cells in the subject, and (a) the abundance of the DNA encoding the CAR reaches at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 1,000 copies per microgram of PBMC genomic DNA for at least 5 days; and / or (b) the population of genetically modified immune cells is established in the subject at a concentration of at least 100 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 5 days.

[0081] As used herein, and unless otherwise specifically stated or understood from the context, a statement that a therapeutic method “does not include administering to the subject a ... preconditioning regimen” refers to therapeutic methods that do not utilize a preconditioning regimen in connection with the therapeutic method itself. For the avoidance of doubt, a cell- therapeutic method of the disclosure that “does not include administering to the subject a ... preconditioning regimen” would still encompass a method of treating a subject that previously received some preconditioning agent (e.g., cyclophosphamide) in connection with a different, non-cell-therapy purpose (e.g., using the agent itself to treat cancer or an autoimmune disease), provided that the subject is not administered a preconditioning agent in preparation for or in connection with the cell therapy itself. Likewise, as used herein, and unless otherwise specifically stated or understood from the context, a statement that a therapeutic method “does not include administering to the subject a purified cytokine or a cell engineered to ectopically express a cytokine” refers to therapeutic methods that do not utilize the administration of such a recombinant cytokine or cell in connection with the therapeutic method itself. For example, a cell therapeutic method of the disclosure that “does not include administering to the subject a purified cytokine” would still encompass methods of treating a subject that previously received such a purified cytokine in connection with a different treatment or purpose (e.g., in connection with treating cancer), provided that the subject is not administered the purified cytokine in preparation for or in connection with the therapeutic method itself.A, Components of Lymphodepleting Preconditioning Regimens

[0082] Lymphodepleting preconditioning regimens are regarded in the art as being an essential step in the use of adoptive CAR therapies to treat diseases, including cancer and autoimmune 1diseases. Lymphodepleting preconditioning is considered in the art to enhance survival, persistence, and efficacy of the administered CAR immune cells, for example, by increasing levels of homeostatic cytokines such as IL-2, IL-7, and IL- 15, and by eliminating endogenous immune cells competing for said cytokines, thereby creating a more favorable environment for the infused cells to proliferate and persist. Lymphodepletion is also considered in the art to be important for reducing the number of cancerous or pathogenic cells (e.g., autoantibodyproducing B cells) prior to commencing CAR cell therapy. Various lymphodepletion preconditioning protocols are known and used in the art. For example, chemotherapeutic agents are commonly used as components in lymphodepleting preconditioning, particularly cyclophosphamide and / or fludarabine. Other chemotherapeutic agents that can be used as components of a preconditioning regimen include bendamustine, oxaliplatin, busulfan, etoposide, and cytarabine. Radiation therapy (e.g., total body irradiation) may also be used in a lymphodepleting preconditioning regimen.

[0083] In spite of the perceived importance of lymphodepletion in the art for treating autoimmune diseases with cell therapies, it has surprisingly now been discovered that lymphodepleting preconditioning regimens are not necessary for treating autoimmune diseases with CAR immune cell therapy. Accordingly, in certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering a lymphodepleting preconditioning regimen to the subject. In certain embodiments, the subject is not administered a lymphodepleting preconditioning regimen for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered a lymphodepleting preconditioning regimen prior to administration of the genetically modified immune cell. In certain embodiments, a lymphodepleting preconditioning regimen is not administered concurrently with the genetically modified immune cell.

[0084] In certain embodiments, a therapeutic method of the disclosure does not include administering a preconditioning regimen comprising administering a chemotherapeutic agent to the subject. In certain embodiments, a therapeutic method of the disclosure does not includeadministering a chemotherapeutic agent to the subject. In certain embodiments, the subject is not administered a chemotherapeutic agent for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered a chemotherapeutic agent prior to administration of the genetically modified immune cell. In certain embodiments, a chemotherapeutic agent is not administered concurrently with the genetically modified immune cell.

[0085] Cyclophosphamide is a prodrug which has been used in the treatment of various diseases, disorders, and conditioning regimens for treating cancer and autoimmune diseases. Following administration to a subject, cyclophosphamide is understood to be activated by the hepatic cytochrome P-450 enzyme system, thereby forming the intermediate metabolite 4- hydroxy cyclophosphamide and its tautomer, aldophosphamide. These two intermediate metabolites may diffuse into cells, where aldophosphamide is subsequently decomposed. In certain instances, aldophosphamide is converted via P-elimination into two molecules, the cytotoxic metabolite phosphoramide mustard and the byproduct acrolein. Alternatively, aldophosphamide can be converted into the inactive, non-toxic metabolite carboxyphosphamide by the activity of aldehyde dehydrogenase (ALDH) enzymes. Accordingly, formation of nontoxic carboxyphosphamide is more likely to occur in cells with high levels of ALDH, such as hematopoietic stem cells and liver cells, whereas formation of the toxic metabolites phosphoramide mustard and acrolein is more likely to occur in cells with low levels of ALDH, such as mature B cells and T cells. Phosphoramide mustard is a bifunctional DNA alkylating molecule which is understood to react with, for example, the N? atom within the imidazole ring of DNA guanine nucleobases, thereby forming covalent linkages between, e.g., two guanine residues on the same strand of DNA (an “intrastrand crosslinkage”) or between two guanine residues on different strands of DNA (an “interstrand crosslinkage”). Formation of these DNA crosslinkages interferes with DNA replication, thereby leading to cell death. The acrolein byproduct of phosphoramide mustard formation is also toxic, and can cause hemorrhagic cystitis (Emadi etal. (2009) NAT. REV. CLIN. ONCOL. 6: 638-647).

[0086] Cyclophosphamide has been used in the treatment of a variety of indications, including cancers such as lymphoma, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma,ovarian cancer, eye cancer, and breast cancer. Cyclophosphamide has also been used in the treatment of certain autoimmune diseases, and in conditioning treatments for allogeneic bone marrow transplantation. Cyclophosphamide is commonly administered to subjects as a component of certain lymphodepleting preconditioning regimens to condition subjects for adoptive cell therapy, e.g., CAR T therapies for the treatment of cancer or autoimmune disease.

[0087] In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering cyclophosphamide to the subject, for example, wherein the method does not include administering a preconditioning regimen comprising administering cyclophosphamide to the subject. In certain embodiments, the subject is not administered cyclophosphamide for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered cyclophosphamide prior to administration of the genetically modified immune cell. In certain embodiments, cyclophosphamide is not administered concurrently with the genetically modified immune cell.

[0088] Fludarabine is a purine analog which differs from physiologic nucleosides in that the sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is generally used in its 5-O-phosphorylated form, i.e., fludarabine phosphate. The term “fludarabine” as used herein is understood to encompass both the non-phosphorylated form and the 5-O-phosphorylated form. Fludarabine acts as a purine antagonist antimetabolite. Once administered to a subject, fludarabine is understood to be dephosphorylated to 2-fluoro-ara-A and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. This metabolite then interferes with DNA replication, likely by inhibiting, e.g., DNA polymerase and ribonucleotide reductase, thus inhibiting DNA synthesis (Gandhi and Plunkett (2002) CLIN. PHARMACOKINET. 41: 93-103). As a result, fludarabine administration leads to increased cell death in dividing cells. Fludarabine is used in the treatment of various diseases and disorders, including hematological malignancies such as lymphomas and leukemias. Fludarabine has also been used in conditioning treatments administered to patients prior to allogeneic stem cell transplantation or adoptive cell therapy. In the context of adoptive T-cell therapy for thetreatment of cancer, the use of fludarabine in lymphodepleting preconditioning regimens has previously been shown to improve T-cell efficacy and persistence and to improve event-free survival of cancer patients (Hay etal. (2019) BLOOD 133(15): 1652-1663; Ramachandran et al. (2019) J. IMMUNOTHER. CANCER 7: 276). Fludarabine is commonly used as a component of lymphodepleting preconditioning regimens administered to patients prior to adoptive cell therapy, e.g., CAR immune cell therapy for the treatment of cancer or autoimmune diseases.

[0089] In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering fludarabine to the subject, for example, wherein the method does not include administering a preconditioning regimen comprising administering fludarabine to the subject. In certain embodiments, the subject is not administered fludarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered fludarabine prior to administration of the genetically modified immune cell. In certain embodiments, fludarabine is not administered concurrently with the genetically modified immune cell.

[0090] In certain embodiments, a therapeutic method of the disclosure does not include administering either cyclophosphamide or fludarabine to the subject. For example, in certain embodiments, the method does not include administering a preconditioning regimen comprising administering either cyclophosphamide or fludarabine to the subject. In certain embodiments, the subject is not administered either cyclophosphamide or fludarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered either cyclophosphamide or fludarabine prior to administration of the genetically modified immune cell. In certain embodiments, neither cyclophosphamide nor fludarabine are administered concurrently with the genetically modified immune cell.

[0091] Bendamustine is an alkylating agent which can be used in the treatment of nonHodgkin’s lymphoma, multiple myeloma, and chronic lymphocytic leukemia (Cheson et al. (2009) J. CLIN. ONCOL. 27(9): 1492-1501). Bendamustine has also been used as a component in lymphodepleting preconditioning regimens prior to CAR T cell therapy for treating cancer. In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering bendamustine to the subject, for example, wherein the method does not include administering a preconditioning regimen comprising administering bendamustine to the subject. In certain embodiments, the method does not include administering bendamustine to the subject. In certain embodiments, the subject is not administered bendamustine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered bendamustine prior to administration of the genetically modified immune cell. In certain embodiments, bendamustine is not administered concurrently with the genetically modified immune cell.

[0092] Oxaliplatin, a chemotherapeutic agent known to damage DNA via the formation of DNA adducts, can be used in the treatment of cancers such as colorectal cancer (Raymond et al. (1998) ANN. ONCOL. 9(10): 1051-73). Oxaliplatin has also been used in combination with cyclophosphamide as a component of lymphodepleting preconditioning prior to CAR T cell therapy for the treatment of cancer (Srivastava etal. (2021) CANCER CELL. 39(2): 193-208). In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering oxaliplatin to the subject. For example, in certain embodiments, the method does not include administering a preconditioning regimen comprising administering oxaliplatin to the subject. In certain embodiments, the subject is not administered oxaliplatin for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered oxaliplatin prior to administration of the genetically modified immune cell. In certainembodiments, oxaliplatin is not administered concurrently with the genetically modified immune cell.

[0093] Busulfan is an alkylating agent which can be used in the treatment of cancers such as chronic myeloid leukemia, as well as in combination with fludarabine or other chemotherapeutic agents for hematopoietic stem cell transplant conditioning (Ciurea et al. (2009) BIOL. BLOOD MARROW TRANSPLANT 15: 523-36). More recently, busulfan has also been used in combination with fludarabine as a component in lymphodepleting preconditioning regimens prior to CAR T cell therapy for treating cancer (Wang et al. (2022) CANCER REP. 5(3): el488). In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering busulfan to the subject, for example, wherein the method does not include administering a preconditioning regimen comprising administering busulfan to the subject. In certain embodiments, the subject is not administered busulfan for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered busulfan prior to administration of the genetically modified immune cell. In certain embodiments, busulfan is not administered concurrently with the genetically modified immune cell.

[0094] Etoposide is a cytotoxic agent which induces DNA breaks by interfering with the function of DNA topoisomerase II (Bromberg etal. (2003) J. BIOL. CHEM. 278(9): 7406-12). Etoposide has been used in the treatment of numerous cancers, and recently has been used in combination with cyclophosphamide and fludarabine as a component of a lymphodepleting preconditioning regimen prior to CAR T cell therapy for treating cancer (Bromberg et al. ; ClinicalTrials.gov ID No. NCT05776407). In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering etoposide to the subject, for example, wherein the method does not include administering a preconditioning regimen comprising administering etoposide to the subject. In certain embodiments, the subject is not administered etoposide for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered etoposide prior to administration of the genetically modified immune cell. In certain embodiments, etoposide is not administered concurrently with the genetically modified immune cell.

[0095] Cytarabine, also known as arabinosylcytosine or cytosine arabinoside (ara-C) is a cytidine analog that acts as a pyrimidine antagonist antimetabolite. Cytarabine contains an arabinose sugar moiety instead of ribose or deoxyribose. Cytarabine is converted into a triphosphate form once inside the cell, and thereby becomes cytotoxic. Cytarabine can be used to treat lymphomas and leukemias and, more recently, has been used in combination with cyclophosphamide, fludarabine, and etoposide in a lymphodepleting preconditioning prior to CAR T cell therapy for treating cancer (Chhikara et al. (2010) EXPERT OPIN. DRUG. DELIV. 7(12): 1399-1414; ClinicalTrials.gov ID No. NCT04499573). In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering cytarabine to the subject, for example, wherein the method does not include administering a preconditioning regimen comprising administering cytarabine to the subject. In certain embodiments, the method does not include administering cytarabine to the subject. In certain embodiments, the subject is not administered cytarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered cytarabine prior to administration of the genetically modified immune cell. In certain embodiments, cytarabine is not administered concurrently with the genetically modified immune cell.

[0096] In certain embodiments, a therapeutic method of the disclosure does not include administering any of cyclophosphamide, fludarabine, bendamustine, oxaliplatin, busulfan, etoposide, or cytarabine to the subject. For example, in certain embodiments, a therapeutic method of the disclosure does not include administering a preconditioning regiment comprising administering any of cyclophosphamide, fludarabine, bendamustine, oxaliplatin, busulfan, etoposide, or cytarabine to the subject. In certain embodiments, the subject is not administeredany of cyclophosphamide, fludarabine, bendamustine, oxaliplatin, busulfan, etoposide, or cytarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered any of cyclophosphamide, fludarabine, bendamustine, oxaliplatin, busulfan, etoposide, or cytarabine prior to administration of the genetically modified immune cell. In certain embodiments, none of cyclophosphamide, fludarabine, bendamustine, oxaliplatin, busulfan, etoposide, or cytarabine are administered concurrently with the genetically modified immune cell.

[0097] In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include treating the subject with radiation therapy (e.g., total body irradiation). For example, in certain embodiments, the method does not include administering a preconditioning regimen comprising treating the subject with radiation therapy (e.g., total body irradiation). In certain embodiments, the subject is not treated with radiation therapy (e.g., total body irradiation) for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not treated with radiation therapy (e.g., total body irradiation) prior to administration of the genetically modified immune cell. b. Cytokines and Cytokine Signaling

[0098] Activation of cytokine signaling has also been described in the art as a strategy to enhance persistence of CAR immune cells used in cell therapy, particularly in the context of using CAR immune cell therapy to treat cancer. Such strategies include, for example, administering purified and / or recombinant cytokines (e.g., IL-2, IL-7, or IL-15) to the subject, e.g., concurrently with and / or after administration of the CAR immune cells. Similar strategies include engineering CAR immune cells to ectopically express cytokines (e.g., IL-7 or IL- 15), cytokine receptors (e.g, IL-2Rb / g, IL-7R, or IL-15R), cytokine signaling proteins, or some combination of the foregoing. However, these strategies may be disadvantageous to use in thetreatment of autoimmune diseases. For example, the administration of exogenous cytokines or modifying cells to express cytokines and related proteins could potentially worsen the autoimmune disease through the activation or potentiation of autoreactive T cells. Accordingly, in certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine or (ii) a purified cytokine (e.g., a wildtype, recombinant, or mutant cytokine, or a functional fragment of any of the foregoing) or a cell engineered to ectopically express a cytokine (e.g., a wild- type, recombinant, or mutant cytokine, or a functional fragment of any of the foregoing). In certain embodiments, a purified cytokine is not administered to the subject in either the same composition as the genetically modified immune cell, or in a different composition.

[0099] In certain embodiments of the therapeutic methods of the disclosure, the subject is not administered a purified cytokine (e.g., IL-2, IL-7, or IL- 15) for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered a purified cytokine prior to administration of the genetically modified immune cell. In certain embodiments, a purified cytokine is not administered concurrently with the genetically modified immune cell. In certain embodiments, the subject is not administered a purified cytokine for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year after administration of the genetically modified immune cell. In certain embodiments, the genetically modified immune cell is administered in a pharmaceutical composition, and the pharmaceutical composition does not comprise a purified cytokine.

[0100] In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimencomprising administering either of cyclophosphamide or fludarabine or (ii) a recombinant or mutant cytokine (e.g., a recombinant or mutant IL-2, IL-7, or IL-15) to the subject. In certain embodiments, the subject is not administered a recombinant or mutant cytokine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered a recombinant or mutant cytokine prior to administration of the genetically modified immune cell. In certain embodiments, a recombinant or mutant cytokine is not administered concurrently with the genetically modified immune cell. In certain embodiments, the subject is not administered a recombinant or mutant cytokine for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year after administration of the genetically modified immune cell. In certain embodiments, the genetically modified immune cell is administered in a pharmaceutical composition, and the pharmaceutical composition does not comprise a recombinant or mutant cytokine.

[0101] In certain embodiments, the disclosure provides a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a CAR, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine or (ii) a cell engineered to ectopically express a cytokine (e.g., a recombinant cytokine, a mutant cytokine, and / or a functional fragment of any of the foregoing) to the subject. In certain embodiments, a therapeutic method of the disclosure does not include administering to the subject a cell engineered to ectopically express a cytokine receptor (e.g., a recombinant cytokine receptor, a mutant cytokine receptor, or a functional fragment thereof). Likewise, in certain embodiments, a therapeutic method of the disclosure does not include administering to the subject a cell engineered to ectopically express a cytokine- pathway signaling protein (e.g., JAK or STAT), or a recombinant or mutant cytokine-pathway signaling protein, or a functional fragment of any of the foregoing.

[0102] For the avoidance of doubt, it should be understood that the phrase “a cell engineered to ectopically express a cytokine,” as used herein, does not include a CAR immune cell that experiences an increase in expression of an endogenous cytokine gene as a result of activation of CAR signaling. For example, an anti-CD19-CAR T cell that becomes active in response to the CAR binding CD 19 on the surface of a target cell, and which consequently experiences an increase in expression of an endogenous cytokine, would not be considered “a cell engineered to ectopically express a cytokine” within the meaning of the phrase as is it is used herein. By contrast, a cell that is engineered to express a non-endogenous, mutant, or recombinant cytokine, or a cell engineered to express a cytokine from a heterologous promoter, would be considered “a cell engineered to ectopically express a cytokine,” as used herein. Similarly, it should be understood that the phrases “a cell engineered to ectopically express a cytokine receptor” and “a cell engineered to ectopically express a cytokine-pathway signaling protein,” as used herein, do not include a CAR immune cell that experiences an increase in expression of an endogenous cytokine receptor gene or an endogenous cytokine-pathway signaling gene, respectively, as a result of the CAR binding its target protein.

[0103] In certain embodiments of the therapeutic methods of the disclosure, the genetically modified immune cell is not engineered to ectopically express any recombinant protein, other than the CAR. In certain embodiments of the therapeutic methods of the disclosure, the genetically modified immune cell is not engineered to ectopically express any protein, other than the CAR. In certain embodiments of the therapeutic methods of the disclosure, the only genetic modification made to the genetically modified immune cell prior to it being administering to the subject is the introduction of the DNA or vector encoding the CAR. In certain embodiments, the only genetic modification made to the genetically modified immune cell is the introduction of the DNA or vector encoding the CAR.

[0104] In certain embodiments of the therapeutic methods of the disclosure, the genetically modified immune cell is not transfected with mRNA encoding a recombinant protein prior to being administered to the subject. In certain embodiments, the genetically modified immune cell is not transfected with synthetic or engineered mRNA prior to being administered to the subject. In certain embodiments, the genetically modified immune cell is not transfected with mRNA prior to being administered to the subject.

[0105] In certain embodiments of the therapeutic methods of the disclosure, the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine or (ii) any of the following: a purified cytokine or functional fragment thereof; a recombinant cytokine; a mutant cytokine; a cell engineered to ectopically express a cytokine (e.g., a recombinant or mutant cytokine, and / or a functional fragment thereof); a cell engineered to ectopically express a cytokine receptor (e.g., a recombinant or mutant cytokine receptor, and / or a functional fragment thereof); and a cell engineered to ectopically express a cytokine- pathway signaling protein (e.g., a recombinant or mutant cytokine- pathway signaling protein, and / or a functional fragment thereof). In certain embodiments, the subject is not administered any of the components enumerated in the foregoing list for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year prior to administration of the genetically modified immune cell. In certain embodiments, the subject is not administered any of the components enumerated in the foregoing list prior to administration of the genetically modified immune cell. In certain embodiments, none of the components enumerated in the foregoing list are administered concurrently with the genetically modified immune cell. In certain embodiments, the subject is not administered any of the components enumerated in the foregoing list for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year after administration of the genetically modified immune cell. c. Persistence of CAR Immune Cells

[0106] In certain embodiments, the therapeutic methods described herein permit the establishment of a population of genetically modified immune cells in the subject being treated. Additionally, in certain embodiments, the population of genetically modified immune cells persists in the subject for some period of time after infusion, which can enhance the therapeutic efficacy of the cell therapy. The presence, abundance, or concentration of genetically modified immune cells in the subject can be measured using any suitable technique or method known in the art, including, e.g, by using qPCR to detect copies of DNA and / or vector encoding the CAR in a blood or PBMC sample obtained from the subject. Additionally or alternatively, the presence, abundance, or concentration of genetically modified immune cells in the subject can bemeasured using flow cytometry to quantify the number or proportion of CAR-expressing, genetically modified immune cells in a blood or PBMC sample obtained from the subject. Similarly, persistence of genetically modified immune cells in the subject can be measured by monitoring the concentration of genetically modified immune cells in the subject’s blood or serum over time following infusion of the genetically modified immune cell.

[0107] In certain embodiments, the population of genetically modified immune cells in the subject is measured in the subject by measuring the number of copies of the CAR-encoding DNA (e.g., CAR-encoding vector) per microgram of total genomic DNA in blood or PBMCs harvested from the subject. The abundance of the CAR-encoding DNA can be measured by qPCR and quantified as the number of DNA copies per microgram of genomic DNA. In certain embodiments of the therapeutic methods of the disclosure, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PMBCs harvested from the subject reaches at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, or at least 90,000 copies per microgram of genomic DNA. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PMBCs harvested from the subject reaches at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, or at least 10,000 copies per microgram of genomic DNA. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 1,000, at least 3,000, at least 5,000, or at least 10,000 copies per microgram of genomic DNA by at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 1,000 copies per microgram of genomic DNA by at least 5 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 3,000 copies per microgram of genomic DNA by at least 5 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 4,000 copies per microgram of genomic DNA by at least 5 daysafter infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 5,000 copies per microgram of genomic DNA by at least 5 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 10,000 copies per microgram of genomic DNA by at least 5 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 20,000 copies per microgram of genomic DNA by at least 5 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the abundance of the DNA encoding the CAR (e.g., vector encoding the CAR) in PBMCs harvested from the subject reaches at least 30,000 copies per microgram of genomic DNA by at least 5 days after infusion of the genetically modified immune cell to the subject.

[0108] In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 3 days, e.g., for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 days, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 days, at least 28, or at least 29 days, e.g, at least 14 days. In certain embodiments, the DNA encoding the CAR (e.g, vector encoding the CAR) remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 3 to 30 days, e.g, 3 to 25 days, 3 to 21 days, 3 to 20 days, 3 to 19 days, 3 to 18 days, 3 to 17 days, 3 to 16 days, 3 to 15 days, 3 to 14 days, 3 to 13 days, 3 to 12 days, 3 to 11 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 30 days, 4 to 25 days, 4 to 21 days, 4 to 20 days, 4 to 19 days, 4 to 18 days, 4 to 17 days, 4 to 16 days, 4 to 15 days, 4 to 14 days, 4 to 13 days, 4 to 12 days, 4 to 11 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to30 days, 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to 13 days, 6 to12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days,8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days, 10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 10 to 30 days.

[0109] In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 3 days, e.g., for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days, e.g, at least 14 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 3,000 copies per microgram of genomic31DNA in PBMCs harvested from the subject for 3 to 30 days, e.g., 3 to 25 days, 3 to 21 days, 3 to 20 days, 3 to 19 days, 3 to 18 days, 3 to 17 days, 3 to 16 days, 3 to 15 days, 3 to 14 days, 3 to 13 days, 3 to 12 days, 3 to 11 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 30 days, 4 to 25 days, 4 to 21 days, 4 to 20 days, 4 to 19 days, 4 to 18 days, 4 to 17 days, 4 to 16 days, 4 to 15 days, 4 to 14 days, 4 to 13 days, 4 to 12 days, 4 to 11 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 30 days, 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to 13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days, 10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days. Incertain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 10 to 30 days.

[0110] In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 4,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 3 days, c.g, for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days, e.g, at least 14 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 4,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 3 to 30 days, e.g, 3 to 25 days, 3 to 21 days, 3 to 20 days, 3 to 19 days, 3 to 18 days, 3 to 17 days, 3 to 16 days, 3 to 15 days, 3 to 14 days, 3 to 13 days, 3 to 12 days, 3 to 11 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 30 days, 4 to 25 days, 4 to 21 days, 4 to 20 days, 4 to 19 days, 4 to 18 days, 4 to 17 days, 4 to 16 days, 4 to 15 days, 4 to 14 days, 4 to 13 days, 4 to 12 days, 4 to 11 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 30 days, 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to 13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days, 10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 5 to 30 days.

[0111] In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 3 days, e.g., for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days, e.g, at least 14 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 3 to 30 days, e.g, 3 to 25 days, 3 to 21 days, 3 to 20 days, 3 to 19 days, 3 to 18 days, 3 to 17 days, 3 to 16 days, 3 to 15 days, 3 to 14 days, 3 to 13 days, 3 to 12 days, 3 to 11 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 30 days, 4 to 25 days, 4 to 21 days, 4 to 20 days, 4 to 19 days, 4 to 18 days, 4 to 17 days, 4 to 16 days, 4 to 15 days, 4 to 14 days, 4 to 13 days, 4 to 12 days, 4 to 11 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 30 days, 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to 13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days, 10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 5 to 30 days.

[0112] In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 3 days, e.g., for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days, e.g, at least 14 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 3 to 21 days, e.g, 3 to 20 days, 3 to 19 days, 3 to18 days, 3 to 17 days, 3 to 16 days, 3 to 15 days, 3 to 14 days, 3 to 13 days, 3 to 12 days, 3 to 11 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 21 days, 4 to 20 days, 4 to 19 days, 4 to 18 days, 4 to 17 days, 4 to 16 days, 4 to 15 days, 4 to 14 days, 4 to 13 days, 4 to 12 days, 4 to 11 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to 13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days,10 to 14 days, 10 to 13 days, 10 to 12 days, 10 to 11 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 21 days, 19 to 20 days, or 20 to 21 days. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for 5 to 30 days.

[0113] In certain embodiments, the genetically modified immune cell is administered to the subject at Day 0, and the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 20,000, or at least 30,000 copies per microgram of genomic DNA in blood orPBMC samples harvested from the subject for at least some period of time after administration. For example, in certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 20,000, or at least 30,000 copies per microgram of genomic DNA in blood or PBMC samples harvested from the subject during the period of time at least from Day 5 to Day 29, from Day 5 to Day 28, from Day 5 to Day 27, from Day 5 to Day 26, from Day 5 to Day 25, from Day 5 to Day 24, from Day 5 to Day 23, from Day 5 to Day 22, from Day 5 to Day 21, from Day 5 to Day 20, from Day 5 to Day 19, from Day 5 to Day 18, from Day 5 to Day 17, from Day 5 to Day 16, from Day 5 to Day 15, from Day 5 to Day 14, from Day 5 to Day 13, from Day 5 to Day 12, from Day 5 to Day 11, from Day 5 to Day 10, from Day 5 to Day 9, from Day 5 to Day 8, from Day 5 to Day 7, from Day 5 to Day 6, from Day 6 to Day 29, from Day 6 to Day 28, from Day 6 to Day 27, from Day 6 to Day 26, from Day 6 to Day 25, from Day 6 to Day 24, from Day 6 to Day 23, from Day 6 to Day 22, from Day 6 to Day 21, from Day 6 to Day 20, from Day 6 to Day 19, from Day 6 to Day 18, from Day 6 to Day 17, from Day 6 to Day 16, from Day 6 to Day 15, from Day 6 to Day 14, from Day 6 to Day 13, from Day 6 to Day 12, from Day 6 to Day 11, from Day 6 to Day 10, from Day 6 to Day 9, from Day 6 to Day 8, from Day 6 to Day 7, from Day 7 to Day 29, from Day 7 to Day 28, from Day 7 to Day 27, from Day 7 to Day 26, from Day 7 to Day 25, from Day 7 to Day 24, from Day 7 to Day 23, from Day 7 to Day 22, from Day 7 to Day 21, from Day 7 to Day 20, from Day 7 to Day 19, from Day 7 to Day 18, from Day 7 to Day 17, from Day 7 to Day 16, from Day 7 to Day 15, from Day 7 to Day 14, from Day 7 to Day 13, from Day 7 to Day 12, from Day 7 to Day 11, from Day 7 to Day 10, from Day 7 to Day 9, from Day 7 to Day 8, from Day 8 to Day 29, from Day 8 to Day 28, from Day 8 to Day 27, from Day 8 to Day 26, from Day 8 to Day 25, from Day8 to Day 24, from Day 8 to Day 23, from Day 8 to Day 22, from Day 8 to Day 21, from Day 8 to Day 20, from Day 8 to Day 19, from Day 8 to Day 18, from Day 8 to Day 17, from Day 8 to Day 16, from Day 8 to Day 15, from Day 8 to Day 14, from Day 8 to Day 13, from Day 8 to Day 12, from Day 8 to Day 11 , from Day 8 to Day 10, from Day 8 to Day 9, from Day 9 to Day 29, from Day 9 to Day 28, from Day 9 to Day 27, from Day 9 to Day 26, from Day 9 to Day 25, from Day9 to Day 24, from Day 9 to Day 23, from Day 9 to Day 22, from Day 9 to Day 21 , from Day 9 to Day 20, from Day 9 to Day 19, from Day 9 to Day 18, from Day 9 to Day 17, from Day 9 to Day 16, from Day 9 to Day 15, from Day 9 to Day 14, from Day 9 to Day 13, from Day 9 to Day 12,from Day 9 to Day 11, from Day 9 to Day 10, from Day 10 to Day 29, from Day 10 to Day 28, from Day 10 to Day 27, from Day 10 to Day 26, from Day 10 to Day 25, from Day 10 to Day 24, from Day 10 to Day 23, from Day 10 to Day 22, from Day 10 to Day 21, from Day 10 to Day 20, from Day 10 to Day 19, from Day 10 to Day 18, from Day 10 to Day 17, from Day 10 to Day 16, from Day 10 to Day 15, from Day 10 to Day 14, from Day 10 to Day 13, from Day 10 to Day 12, from Day 10 to Day 11, from Day 11 to Day 29, from Day 11 to Day 28, from Day 11 to Day 27, from Day 11 to Day 26, from Day 11 to Day 25, from Day 11 to Day 24, from Day 11 to Day 23, from Day 11 to Day 22, from Day 11 to Day 21, from Day 11 to Day 20, from Day 11 to Day 19, from Day 11 to Day 18, from Day 11 to Day 17, from Day 11 to Day 16, from Day 11 to Day 15, from Day 11 to Day 14, from Day 11 to Day 13, from Day 11 to Day 12, from Day 12 to Day 29, from Day 12 to Day 28, from Day 12 to Day 27, from Day 12 to Day 26, from Day 12 to Day 25, from Day 12 to Day 24, from Day 12 to Day 23, from Day 12 to Day 22, from Day 12 to Day 21, from Day 12 to Day 20, from Day 12 to Day 19, from Day 12 to Day 18, from Day 12 to Day 17, from Day 12 to Day 16, from Day 12 to Day 15, from Day 12 to Day 14, from Day 12 to Day 13, from Day 13 to Day 29, from Day 13 to Day 28, from Day 13 to Day 27, from Day 13 to Day 26, from Day 13 to Day 25, from Day 13 to Day 24, from Day 13 to Day 23, from Day 13 to Day 22, from Day 13 to Day 21, from Day 13 to Day 20, from Day 13 to Day 19, from Day 13 to Day 18, from Day 13 to Day 17, from Day 13 to Day 16, from Day 13 to Day 15, from Day 13 to Day 14, from Day 14 to Day 29, from Day 14 to Day 28, from Day 14 to Day 27, from Day 14 to Day 26, from Day 14 to Day 25, from Day 14 to Day 24, from Day 14 to Day 23, from Day 14 to Day 22, from Day 14 to Day 21, from Day 14 to Day 20, from Day 14 to Day 19, from Day 14 to Day 18, from Day 14 to Day 17, from Day 14 to Day 16, or at least from Day 14 to Day 15. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 1,000 vector copies per microgram of genomic DNA in blood or PBMC samples harvested from the subject during the period of time at least from Day 5 to Day 14. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 3,000 vector copies per microgram of genomic DNA in blood or PBMC samples harvested from the subject during the period of time at least from Day 5 to Day 14. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 4,000 vector copies per microgram of genomic DNA in blood or PBMC samples harvested from the subject during the period of time at least from Day 5 to Day 14. Incertain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 5,000 vector copies per microgram of genomic DNA in blood or PBMC samples harvested from the subject during the period of time at least from Day 5 to Day 14. In certain embodiments, the DNA encoding the CAR (e.g., vector encoding the CAR) remains at an abundance of at least 10,000 vector copies per microgram of genomic DNA in blood or PBMC samples harvested from the subject during the period of time at least from Day 5 to Day 14.

[0114] In certain embodiments, the population of genetically modified immune cells in the subject is established at a concentration of at least 100 cells per microliter of blood, e.g., as measured by flow cytometry. For example, in certain embodiments, the population of genetically modified immune cells is established in the subject at a concentration of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, or at least 10,000 cells per microliter of blood. In certain embodiments, the population of genetically modified immune cells is established in the subject at a concentration of at least 100, at least 200, at least 300, or at least 1,000 cells per microliter of blood. In certain embodiments, the population of genetically modified immune cells is established in the subject at a concentration of at least 100, at least 200, at least 300, or at least 1,000 cells per microliter of blood by at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or at least 14 days after infusion of the genetically modified immune cell to the subject. In certain embodiments, the population of genetically modified immune cells is established in the subject at a concentration of at least 100, at least 200, at least 300 cells, or at least 1,000 per microliter of blood by at least 5 days after infusion of the genetically modified immune cell to the subject.

[0115] In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 5 days, e.g., for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days, e.g., at least 5 days. In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of bloodfor 5 to 30 days, e.g., 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days,10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days.

[0116] In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 200 cells per microliter of blood for at least 5 days, e.g., for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days, e.g., at least 5 days. In certain embodiments, the population of genetically modifiedimmune cells persists in the subject at a concentration of at least 200 cells per microliter of blood for 5 to 30 days, e.g., 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days,10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days.

[0117] In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood for at least 5 days, e.g., for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21days, e.g., at least 5 days. In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood for 5 to 30 days, e.g., 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to 19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days,10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days.

[0118] In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 1,000 cells per microliter of blood for at least 5 days, e.g., for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days, e.g., at least 5 days. In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 1,000 cells per microliter of blood for 5 to 30 days, e.g., 5 to 25 days, 5 to 21 days, 5 to 20 days, 5 to 19 days, 5 to 18 days, 5 to 17 days, 5 to 16 days, 5 to 15 days, 5 to 14 days, 5 to 13 days, 5 to 12 days, 5 to 11 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 30 days, 6 to 25 days, 6 to 21 days, 6 to 20 days, 6 to 19 days, 6 to 18 days, 6 to 17 days, 6 to 16 days, 6 to 15 days, 6 to 14 days, 6 to 13 days, 6 to 12 days, 6 to 11 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 30 days, 7 to 25 days, 7 to 21 days, 7 to 20 days, 7 to 19 days, 7 to 18 days, 7 to 17 days, 7 to 16 days, 7 to 15 days, 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 30 days, 8 to 25 days, 8 to 21 days, 8 to 20 days, 8 to 19 days, 8 to 18 days, 8 to 17 days, 8 to 16 days, 8 to 15 days, 8 to 14 days, 8 to 13 days, 8 to 12 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 30 days, 9 to 25 days, 9 to 21 days, 9 to 20 days, 9 to19 days, 9 to 18 days, 9 to 17 days, 9 to 16 days, 9 to 15 days, 9 to 14 days, 9 to 13 days, 9 to 12 days, 9 to 11 days, 9 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 21 days, 10 to 20 days, 10 to 19 days, 10 to 18 days, 10 to 17 days, 10 to 16 days, 10 to 15 days, 10 to 14 days, 10 to 13 days,10 to 12 days, 10 to 11 days, 11 to 30 days, 11 to 25 days, 11 to 21 days, 11 to 20 days, 11 to 19 days, 11 to 18 days, 11 to 17 days, 11 to 16 days, 11 to 15 days, 11 to 14 days, 11 to 13 days, 11 to 12 days, 12 to 30 days, 12 to 25 days, 12 to 21 days, 12 to 20 days, 12 to 19 days, 12 to 18 days, 12 to 17 days, 12 to 16 days, 12 to 15 days, 12 to 14 days, 12 to 13 days, 13 to 30 days, 13 to 25 days, 13 to 21 days, 13 to 20 days, 13 to 19 days, 13 to 18 days, 13 to 17 days, 13 to 16 days, 13 to 15 days, 13 to 14 days, 14 to 30 days, 14 to 25 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, 14 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 21 days, 15 to 20 days, 15 to 19 days, 15 to 18 days, 15 to 17 days, 15 to 16 days, 16 to 30 days, 16 to 25 days, 16 to 21 days, 16 to 20 days, 16 to 19 days, 16 to 18 days, 16 to 17 days, 17 to 30 days, 17 to 25 days, 17 to 21 days, 17 to 20 days, 17 to 19 days, 17 to 18 days, 18 to 30 days, 18 to 25 days, 18 to 21 days, 18 to 20 days, 18 to 19 days, 19 to 30 days, 19 to 25 days, 19 to 21 days, 19 to 20 days, 20 to 30 days, 20 to 25 days, 20 to 21 days, 21 to 30 days, 21 to 25 days, or 25 to 30 days.

[0119] In certain embodiments, the genetically modified immune cell is administered to the subject at Day 0, and the population of genetically modified immune cells persists in the subjectat a concentration of at least 100, at least 200, at least 300, or at least 1,000 cells per microliter of blood for at least some period of time after administration. For example, in certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100, at least 200, at least 300 cells, or at least 1,000 cells per microliter of blood for at least from Day 5 to Day 29, from Day 5 to Day 28, from Day 5 to Day 27, from Day 5 to Day 26, from Day 5 to Day 25, from Day 5 to Day 24, from Day 5 to Day 23, from Day5 to Day 22, from Day 5 to Day 21, from Day 5 to Day 20, from Day 5 to Day 19, from Day 5 to Day 18, from Day 5 to Day 17, from Day 5 to Day 16, from Day 5 to Day 15, from Day 5 to Day 14, from Day 5 to Day 13, from Day 5 to Day 12, from Day 5 to Day 11, from Day 5 to Day 10, from Day 5 to Day 9, from Day 5 to Day 8, from Day 5 to Day 7, from Day 5 to Day 6, from Day 6 to Day 29, from Day 6 to Day 28, from Day 6 to Day 27, from Day 6 to Day 26, from Day6 to Day 25, from Day 6 to Day 24, from Day 6 to Day 23, from Day 6 to Day 22, from Day 6 to Day 21, from Day 6 to Day 20, from Day 6 to Day 19, from Day 6 to Day 18, from Day 6 to Day 17, from Day 6 to Day 16, from Day 6 to Day 15, from Day 6 to Day 14, from Day 6 to Day 13, from Day 6 to Day 12, from Day 6 to Day 11, from Day 6 to Day 10, from Day 6 to Day 9, from Day 6 to Day 8, from Day 6 to Day 7, from Day 7 to Day 29, from Day 7 to Day 28, from Day 7 to Day 27, from Day 7 to Day 26, from Day 7 to Day 25, from Day 7 to Day 24, from Day 7 to Day 23, from Day 7 to Day 22, from Day 7 to Day 21, from Day 7 to Day 20, from Day 7 to Day 19, from Day 7 to Day 18, from Day 7 to Day 17, from Day 7 to Day 16, from Day 7 to Day 15, from Day 7 to Day 14, from Day 7 to Day 13, from Day 7 to Day 12, from Day 7 to Day 11, from Day 7 to Day 10, from Day 7 to Day 9, from Day 7 to Day 8, from Day 8 to Day 29, from Day 8 to Day 28, from Day 8 to Day 27, from Day 8 to Day 26, from Day 8 to Day 25, from Day8 to Day 24, from Day 8 to Day 23, from Day 8 to Day 22, from Day 8 to Day 21, from Day 8 to Day 20, from Day 8 to Day 19, from Day 8 to Day 18, from Day 8 to Day 17, from Day 8 to Day 16, from Day 8 to Day 15, from Day 8 to Day 14, from Day 8 to Day 13, from Day 8 to Day 12, from Day 8 to Day 11 , from Day 8 to Day 10, from Day 8 to Day 9, from Day 9 to Day 29, from Day 9 to Day 28, from Day 9 to Day 27, from Day 9 to Day 26, from Day 9 to Day 25, from Day9 to Day 24, from Day 9 to Day 23, from Day 9 to Day 22, from Day 9 to Day 21 , from Day 9 to Day 20, from Day 9 to Day 19, from Day 9 to Day 18, from Day 9 to Day 17, from Day 9 to Day 16, from Day 9 to Day 15, from Day 9 to Day 14, from Day 9 to Day 13, from Day 9 to Day 12, from Day 9 to Day 11, from Day 9 to Day 10, from Day 10 to Day 29, from Day 10 to Day 28,from Day 10 to Day 27, from Day 10 to Day 26, from Day 10 to Day 25, from Day 10 to Day 24, from Day 10 to Day 23, from Day 10 to Day 22, from Day 10 to Day 21, from Day 10 to Day 20, from Day 10 to Day 19, from Day 10 to Day 18, from Day 10 to Day 17, from Day 10 to Day 16, from Day 10 to Day 15, from Day 10 to Day 14, from Day 10 to Day 13, from Day 10 to Day 12, from Day 10 to Day 11, from Day 11 to Day 29, from Day 11 to Day 28, from Day 11 to Day 27, from Day 11 to Day 26, from Day 11 to Day 25, from Day 11 to Day 24, from Day 11 to Day 23, from Day 11 to Day 22, from Day 11 to Day 21, from Day 11 to Day 20, from Day 11 to Day 19, from Day 11 to Day 18, from Day 11 to Day 17, from Day 11 to Day 16, from Day 11 to Day 15, from Day 11 to Day 14, from Day 11 to Day 13, from Day 11 to Day 12, from Day 12 to Day 29, from Day 12 to Day 28, from Day 12 to Day 27, from Day 12 to Day 26, from Day 12 to Day 25, from Day 12 to Day 24, from Day 12 to Day 23, from Day 12 to Day 22, from Day 12 to Day 21, from Day 12 to Day 20, from Day 12 to Day 19, from Day 12 to Day 18, from Day 12 to Day 17, from Day 12 to Day 16, from Day 12 to Day 15, from Day 12 to Day 14, from Day 12 to Day 13, from Day 13 to Day 29, from Day 13 to Day 28, from Day 13 to Day 27, from Day 13 to Day 26, from Day 13 to Day 25, from Day 13 to Day 24, from Day 13 to Day 23, from Day 13 to Day 22, from Day 13 to Day 21, from Day 13 to Day 20, from Day 13 to Day 19, from Day 13 to Day 18, from Day 13 to Day 17, from Day 13 to Day 16, from Day 13 to Day 15, from Day 13 to Day 14, from Day 14 to Day 29, from Day 14 to Day 28, from Day 14 to Day 27, from Day 14 to Day 26, from Day 14 to Day 25, from Day 14 to Day 24, from Day 14 to Day 23, from Day 14 to Day 22, from Day 14 to Day 21, from Day 14 to Day 20, from Day 14 to Day 19, from Day 14 to Day 18, from Day 14 to Day 17, from Day 14 to Day 16, or at least from Day 14 to Day 15. In certain embodiments, the population of genetically modified immune cells persists in the subject at a concentration of at least 100, at least 200, at least 300, or at least 1,000 cells per microliter of blood for at least from Day 5 to Day 10 or at least from Day 5 to Day 14. d. Cell Therapy

[0120] The therapeutic methods described herein comprise administering a genetically modified immune cell comprising a DNA encoding a CAR to a subject. The immune cell can be, e.g., a T cell or an NK cell. In certain embodiments, the genetically modified immune cell is administered in an immune cell composition. In certain embodiments, the immune cell composition comprises NK cells. In certain embodiments, the immune cell composition comprises T cells, e.g., CD8+ T cells. In certain embodiments, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the immune cell composition are CD8+ T cells. In certain embodiments, the immune cell composition further comprises CD4+ T cells, optionally wherein at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells in the immune cell composition are CD4+ T cells.

[0121] Immune cells for use in immune cell therapy can be obtained from a blood sample of a patient by apheresis. In certain embodiments, a lymphocyte-rich fraction and a monocyte-rich fraction can be acquired by elutriating peripheral blood mononuclear cells (PBMCs) of the patient. The monocyte-rich fraction can then be used to prepare antigen-presenting cells (APCs) for priming T cells, which can be obtained from the lymphocyte-rich fraction. Exemplary methods are described in W02020055931 and U.S. Patent Nos. 9,963,677 and 9,642,906. In certain embodiments, the immune cell therapy is autologous, i.e., immune cells obtained from a patient, after in vitro culture, are administered to the same patient. In certain embodiments, the immune cell therapy is allogeneic, optionally wherein the immune cells are genetically engineered to abrogate expression via component inactivation of class I MHC (e.g., 02M), class II (e.g., RFXANK), or TCR (e.g. TRAC, or CD3).

[0122] In certain embodiments, the immune cell for use in cell therapy is a T cell, e.g, a cytotoxic T cell, a helper T cell, a memory T cell, a regulatory T cell, a gamma delta T cell, a natural killer cell, a cytokine induced killer cell, a cell line thereof, a T memory stem cell, or another T effector cell. In certain embodiments, the T cell specifically targets a cell expressing the targeted protein, e.g, a B cell expressing CD 19, or a B cell expressing an autoantibody on its surface (e.g., an anti-MuSK antibody). In certain embodiments, the T cell is autologous to the subject. In other embodiments, the T cell is allogeneic to the subject.

[0123] Immune cells (e.g., T cells, e.g, CD8+T cells) can have antigen specificity via a receptor expressed on the cell surface. T cells can naturally express T cell receptors (TCRs), such as aPTCRs or ySTCRs, and can be “trained” to express TCRs that target a given antigen or epitope. Immune cells can also be genetically engineered to express a recombinant TCR or a chimeric antigen receptor (CAR). A CAR for use in the therapeutic methods disclosed can target one or more antigens or epitopes thereof present on an immune cell surface. In certain embodiments, the immune cells are genetically engineered to inactivate their endogenous TCRs,for example, by knocking out the TRAC or TRBC gene, to reduce ligand-independent tonic T cell signaling and enhances T cell potency.

[0124] The immune cells for use in immune cell therapy can be genetically engineered to express a CAR by introducing a nucleic acid encoding the CAR. In certain embodiments, the nucleic acid is a DNA molecule (e.g., a cDNA molecule). In certain embodiments, the nucleic acid further comprises an expression control sequence (e.g., promoter and / or enhancer) operably linked to the CAR coding sequence. In certain embodiments, the immune cells are transduced by a vector, such as a viral vector (e.g., AAV vector, lenti viral vector, or adenoviral vector) or a non-viral vector (e.g., plasmid), comprising a nucleic acid encoding the CAR. In certain embodiments, the nucleic acid encodes an amino acid sequence comprising a signal peptide at the N-terminus of the CAR. Such a signal peptide can facilitate cell surface localization of the CAR when it is expressed in an effector cell, and is cleaved from the CAR during cellular processing.

[0125] In certain embodiments, the therapeutic methods of the disclosure comprise administering a dose of genetically modified immune cells (e.g., CAR T cells) to a subject in need thereof. In certain embodiments, the dose of genetically modified immune cells is from 1x105cells / kg bodyweight to 1x108cells / kg body weight. For example, in certain embodiments, the dose of genetically modified immune cells is from 1x105to 1x108cells / kg, from 1x105to 9xl07cells / kg, from IxlO5to 6xl07cells / kg, from IxlO5to 3xl07cells / kg, from IxlO5to IxlO7cells / kg, from IxlO5to 9xl06cells / kg, from IxlO5to 8xl06cells / kg, from IxlO5to 7xl06cells / kg, from IxlO5to 6xl06cells / kg, from IxlO5to 5xl06cells / kg, from IxlO5to 4xl06cells / kg, from IxlO5to 3xl06cells / kg, from IxlO5to 2xl06cells / kg, from IxlO5to IxlO6cells / kg, from IxlO5to 9xl05cells / kg, from IxlO5to 6xl05cells / kg, from IxlO5to 3xlO5cells / kg, from 3x105to IxlO8cells / kg, from 3x105to 9x107cells / kg, from 3x105to 6x107cells / kg, from 3x105to 3x107cells / kg, from 3x105to IxlO7cells / kg, from 3x105to 9x106cells / kg, from 3x105to 8x106cells / kg, from 3x105to 7x106cells / kg, from 3x105to 6x106cells / kg, from 3x105to 5x106cells / kg, from 3x105to 4x106cells / kg, from 3x105to 3x106cells / kg, from 3x105to 2x106cells / kg, from 3x105to IxlO6cells / kg, from 3x105to 9x105cells / kg, from 3x105to 6x105cells / kg, from 6x105to IxlO8cells / kg, from 6x105to 9x107cells / kg, from 6x105to 6x107cells / kg, from 6x105to 3x107cells / kg, from 6x105to IxlO7cells / kg, from 6x105to 9x106cells / kg, from 6x105to 8x106cells / kg, from 6x105to 7x106cells / kg, from 6x105to 6x106cells / kg, from 6x105to 5x106cells / kg, from 6x105to 4x106cells / kg, from 6x105to 3x106cells / kg, from 6x105to 2x106cells / kg, from 6x105to 1x106cells / kg, from 6x105to 9x105cells / kg, from 9x105to 1x108cells / kg, from 9x105to 9x107cells / kg, from 9x105to 6x107cells / kg, from 9x105to 3x107cells / kg, from 9x105to 1x107cells / kg, from 9x105to 9x106cells / kg, from 9x105to 8x106cells / kg, from 9x105to 7x106cells / kg, from 9x105to 6x106cells / kg, from 9x105to 5x106cells / kg, from 9x105to 4x106cells / kg, from 9x105to 3x106cells / kg, from 9x105to 2x106cells / kg, from 9x105to 1x106cells / kg, from IxlO6to IxlO8cells / kg, from IxlO6to 9xl07cells / kg, from IxlO6to 6xl07cells / kg, from IxlO6to 3xl07cells / kg, from IxlO6to IxlO7cells / kg, from IxlO6to 9xl06cells / kg, from IxlO6to 8xl06cells / kg, from IxlO6to 7xl06cells / kg, from IxlO6to 6xl06cells / kg, from IxlO6to 5xl06cells / kg, from IxlO6to 4xl06cells / kg, from IxlO6to 3xl06cells / kg, from IxlO6to 2x106cells / kg, from 2x106to IxlO8cells / kg, from 2x106to 9x107cells / kg, from 2x106to 6x107cells / kg, from 2x106to 3x107cells / kg, from 2x106to IxlO7cells / kg, from 2x106to 9x106cells / kg, from 2x106to 8x106cells / kg, from 2x106to 7x106cells / kg, from 2x106to 6x106cells / kg, from 2x106to 5x106cells / kg, from 2x106to 4x106cells / kg, from 2x106to 3x106cells / kg, from 3x106to IxlO8cells / kg, from 3x106to 9x107cells / kg, from 3x106to 6x107cells / kg, from 3x106to 3x107cells / kg, from 3x106to IxlO7cells / kg, from 3x106to 9x106cells / kg, from 3x106to 8x106cells / kg, from 3x106to 7x106cells / kg, from 3x106to 6x106cells / kg, from 3x106to 5x106cells / kg, from 3x106to 4x106cells / kg, from 4x106to IxlO8cells / kg, from 4x106to 9x107cells / kg, from 4x106to 6x107cells / kg, from 4x106to 3x107cells / kg, from 4x106to IxlO7cells / kg, from 4x106to 9x106cells / kg, from 4x106to 8x106cells / kg, from 4x106to 7x106cells / kg, from 4x106to 6x106cells / kg, from 4x106to 5x106cells / kg, from 5x106to IxlO8cells / kg, from 5x106to 9x107cells / kg, from 5x106to 6x107cells / kg, from 5x106to 3x107cells / kg, from 5x106to IxlO7cells / kg, from 5x106to 9x106cells / kg, from 5x106to 8x106cells / kg, from 5x106to 7x106cells / kg, from 5x106to 6x106cells / kg, from 6x106to IxlO8cells / kg, from 6x106to 9x107cells / kg, from 6x106to 6x107cells / kg, from 6x106to 3x107cells / kg, from 6x106to IxlO7cells / kg, from 6x106to 9x106cells / kg, from 6x106to 8x106cells / kg, from 6x106to 7x106cells / kg, from 7x106to IxlO8cells / kg, from 7x106to 9x107cells / kg, from 7x106to 6x107cells / kg, from 7x106to 3x107cells / kg, from 7x106to IxlO7cells / kg, from 7x106to 9x106cells / kg, from 7x106to 8x106cells / kg, from 8x106to IxlO8cells / kg, from 8x106to 9x107cells / kg, from 8x106to 6x107cells / kg, from 8x106to 3x107cells / kg, from 8x106to 1x107cells / kg, from 8x106to 9x106cells / kg, from 9x106to 1x108cells / kg, from 9x106to 9x107cells / kg, from 9x106to 6x107cells / kg, from 9x106to 3x107cells / kg, from 9x106to 1x107cells / kg, from IxlO7to IxlO8cells / kg, from IxlO7to 9xl07cells / kg, from IxlO7to 6xl07cells / kg, from IxlO7to 3x107cells / kg, from 3x107to IxlO8cells / kg, from 3x107to 9x107cells / kg, from 3x107to 6x107cells / kg, from 6x107to IxlO8cells / kg, from 6x107to 9x107cells / kg, or from 9x107to IxlO8cells / kg. In certain embodiments, the dose of genetically modified immune cells is from IxlO6to IxlO7cells / kg. In certain embodiments, the dose of genetically modified immune cells is an effective amount of the genetically modified immune cells.

[0126] In certain embodiments, the dose of genetically modified immune cells is at least IxlO5cells / kg. For example, in certain embodiments, the dose of genetically modified immune cells is at least IxlO8cells / kg, at least 9xl07cells / kg, at least 6xl07cells / kg, at least 3xl07cells / kg, at least IxlO7cells / kg, at least 9x106cells / kg, at least 8x106cells / kg, at least 7x106cells / kg, at least 6x106cells / kg, at least 5x106cells / kg, at least 4x106cells / kg, at least 3x106cells / kg, at least 2x106cells / kg, at least IxlO6cells / kg, at least 9x105cells / kg, at least 6x105cells / kg, at least 3x105cells / kg, or at least IxlO5cells / kg. In certain embodiments, the dose of immune cells is at least IxlO6cells / kg. In certain embodiments, the dose of immune cells is IxlO8cells / kg, 9x107cells / kg, 6x107cells / kg, 3x107cells / kg, IxlO7cells / kg, 9x106cells / kg, 8x106cells / kg, 7x106cells / kg, 6x106cells / kg, 5x106cells / kg, 4x106cells / kg, 3x106cells / kg, 2x106cells / kg, IxlO6cells / kg, 9x105cells / kg, 6x105cells / kg, 3x105cells / kg, or IxlO5cells / kg. In certain embodiments, the dose of immune cells is IxlO6cells / kg. In certain embodiments, the dose of immune cells is 3x106cells / kg. In certain embodiments, the dose of immune cells is IxlO7cells / kg.

[0127] In certain embodiments, the total dose of genetically modified immune cells (e.g., CAR T cells) administered to the subject is from IxlO6to IxlO10cells. For example, in certain embodiments, the total dose of genetically modified immune cells administered to the subject is from IxlO6to IxlO10cells, from IxlO6to 5xl09cells, from IxlO6to IxlO9cells, from IxlO6to 5xl08cells, from IxlO6to IxlO8cells, from IxlO6to 5xl07cells, from IxlO6to IxlO7cells, from IxlO6to 5xl06cells, from 5xl06to IxlO10cells, from 5xl06to 5xl09cells, from 5xl06to IxlO9cells, from 5x106to 5x108cells, from 5x106to IxlO8cells, from 5x106to 5x107cells,from 5x106to 1x107cells, from 1x107to 1x1010cells, from 1x107to 5x109cells, from 1x107to IxlO9cells, from IxlO7to 5xl08cells, from IxlO7to IxlO8cells, from IxlO7to 5xl07cells, from 5x107to IxlO10cells, from 5x107to 5x109cells, from 5x107to IxlO9cells, from 5x107to 5xl08cells, from 5xl07to IxlO8cells, from IxlO8to IxlO10cells, from IxlO8to 5xl09cells, from IxlO8to IxlO9cells, from IxlO8to 5xl08cells, from 5xl08to IxlO10cells, from 5xl08to 5xl09cells, from 5xl08to IxlO9cells, from IxlO9to IxlO10cells, from IxlO9to 5xl09cells, or from 5x109to IxlO10cells.

[0128] In certain embodiments, the total dose of genetically modified immune cells (e.g., CAR T cells) administered to the subject is at least IxlO6cells. For example, in certain embodiments, the total dose of genetically modified immune cells administered to the subject is at least IxlO6cells, at least 2.5xl06cells, at least 5x106cells, at least 7.5xl06cells, at least IxlO7cells, at least 2.5xl07cells, at least 5x107cells, at least 7.5xl07cells, at least IxlO8cells, at least 2.5xl08cells, at least 5x108cells, at least 7.5xl08cells, at least IxlO9cells, at least 2.5xl09cells, at least 5x109cells, at least 7.5xl09cells, or at least IxlO10cells.

[0129] In certain embodiments, the immune cells are administered to the subject in a single dose. In certain embodiments, the genetically modified immune cell (or the dose of genetically modified immune cells) is administered intravenously.

[0130] The disclosure further relates to pharmaceutical compositions comprising the genetically modified immune cell (e.g., CAR T cells) as described herein for use in the therapeutic methods of the disclosure. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. e. Therapeutic Uses

[0131] The present disclosure provides methods for decreasing an unwanted immune response in a subject by administering a genetically modified immune cell (e.g., a CAR T cell), wherein the immune cell targets and kills a target immune cell (e.g., a B cell), thereby reducing the unwanted immune response in the subject.

[0132] In certain instances, unwanted immune responses are mediated, at least in part, by the activity of immunoglobulins. Immunoglobulins are glycoproteins belonging to the immunoglobulin superfamily which recognize antigens and facilitate the humoral response of theimmune system. Immunoglobulins may occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell and is referred to as the B cell receptor (BCR). Immature B cells, which have not been exposed to an antigen, are known as naive B cells and express only the IgM isotype in a cell surface-bound form. B cells begin to express both IgM and IgD when they reach maturity, indicating that they are ready to respond to antigen. B cell activation follows engagement of the BCR with an antigen, causing the cell to divide and differentiate into an antibody-producing plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. However, B cells producing antibodies directed against self antigens (autoantibodies) can lead to an unwanted immune response against an organism’s own cells and tissues, thereby contributing to an autoimmune disorder.

[0133] Thus, the disclosure provides a method of treating an autoimmune disease in a subject. The method comprises administering to the subject a genetically modified CAR immune cell (e.g., a CAR T cell) which targets an immune cell, e.g, which targets a B cell. In certain embodiments, the method is used to decrease the number of circulating B cells in a subject. In certain embodiments, B cells in the subject produce autoantibodies having specificity for self antigens, e.g, anti-dsDNA antibodies or anti-MuSK antibodies. In certain embodiments, the method is used to decrease the number of antibodies, e.g, autoantibodies, in a subject. In certain embodiments, the autoimmune disease is a B-cell-mediated autoimmune disease, i.e., an autoimmune disease wherein (1) at least some symptoms or manifestations of the disease are attributable to, and / or exacerbated by, immunoglobulins produced by the subject’s B cells, and / or (2) B cells have some role in initiating or maintaining the disease or a symptom or manifestation thereof.

[0134] Examples of B-cell-mediated autoimmune diseases include, but are not limited to: systemic lupus erythematosus (SLE), lupus nephritis, SLE with anti-dsDNA antibodies, pemphigus vulgaris (PV), mucosal PV, mucocutaneous PV, myasthenia gravis (MG), MuSK- associated MG, AChR MG, myositis, (e.g., juvenile myositis), membranous nephropathy, antisynthetase syndrome, dermatomyositis, immune mediated necrotizing myopathy, multiple sclerosis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, anti- NMDA Receptor encephalitis, Lambert-Eaton syndrome, pemphigus foliaceus, epidermolysis bullosa acquisita, bullous pemphigoid, Goodpasture’s syndrome, rheumatoid arthritis, systemicsclerosis, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, immune thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, type 1 diabetes, Grave’s disease, and Hashimoto’s disease. In certain embodiments, a therapeutic method described herein is used to treat any of the foregoing autoimmune diseases in a subject. In certain embodiments, a therapeutic method described herein is used to treat SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK- associated MG, AChR MG, myositis (e.g., juvenile myositis), systemic sclerosis, chronic immune demyelinating polyneuropathy, membranous nephropathy, anti-synthetase syndrome, dermatomyositis, or immune mediated necrotizing myopathy in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat SLE, PV, MG, myositis, juvenile myositis, systemic sclerosis, multiple sclerosis, membranous nephropathy, or chronic immune demyelinating polyneuropathy in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK-associated MG, AChR MG, myositis, membranous nephropathy, anti-synthetase syndrome, dermatomyositis, juvenile myositis, or immune mediated necrotizing myopathy in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat SLE in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat myasthenia gravis (e.g., MuSK myasthenia gravis) in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat pemphigus gravis in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat lupus nephritis in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat idiopathic inflammatory myopathy (e.g., juvenile idiopathic inflammatory myopathy) in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat systemic sclerosis in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat scleroderma in a subject in need thereof. In certain embodiments, a therapeutic method described herein is used to treat generalized myasthenia gravis (gMG) in a subject in need thereof.

[0135] In certain embodiments, treating a subject using a method of the disclosure results in a decrease in autoantibody levels (e.g., anti-dsDNA antibody levels, anti-nuclear antibody levels,or anti-MuSK antibody levels) in the subject. For example, in certain embodiments, autoantibody levels in the subject decrease by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% e.g., as compared to autoantibody levels in the subject prior to administering the treatment. In certain embodiments, treating a subject with a method of the disclosure makes autoantibody levels undetectable in the subject. Autoantibody levels can be measured using any appropriate technique known in the art including, for example, by ELISA.

[0136] In certain embodiments, treating a subject using a method of the disclosure results in a decrease in circulating B cell levels. For example, in certain embodiments, B cell levels in the subject decrease by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, e.g., as compared to levels of circulating B cells in the subject prior to administering the treatment. In certain embodiments, treating a subject with a method of the disclosure makes circulating B cell levels undetectable in the subject. In certain embodiments, the decrease in circulating B cells is transient, whereby B cell levels are restored partially or completely after a period of time.

[0137] In certain embodiments, treating a subject using a method of the disclosure results in a decrease in the amount of circulating CD19+ cells. For example, in certain embodiments, the amount of circulating CD19+ cells in the subject decreases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, e.g., as compared to levels of circulating B cells in the subject prior to administering the treatment. In certain embodiments, treating a subject with a method of the disclosure makes the amount of circulating CD 19+ cells undetectable in the subject. In certain embodiments, the decrease in the amount of circulating CD 19+ cells is transient, and levels are restored partially or completely after a period of time.

[0138] In certain embodiments, therapeutic methods of the disclosure may be used to treat SLE in a subject.

[0139] In certain embodiments, treating lupus nephritis in a subject using a method of the disclosure results in a reduction in proteinuria, e.g., as measured by urine protein concentration. For example, in certain embodiments, proteinuria in the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, e.g., as compared to proteinuria in the subject prior to administering the treatment or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, treating a subject with a method of the disclosure eliminates proteinuria.

[0140] In certain embodiments, treating SLE in a subject using a method of the disclosure results in an increase in the amount or activity of one or more complement factors, e.g., as detected by measuring serum levels of complement component 3 (C3) or complement component 4 (C4), or by measuring the 50% hemolytic complement (CH50) activity. For example, in certain embodiments, the amount of a complement factor in a subject is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%, e.g., as compared to the amount of the complement factor in the subject prior to administering the treatment, or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, the CH50 activity in a subject is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%, e.g., as compared to the CH50 activity in the subject prior to administering the treatment, or as compared to an appropriate control subject or group that did not receive the treatment.

[0141] In certain embodiments, treating an autoimmune disease in a subject using a method of the disclosure results in a decrease in a disease symptom or disease activity. For example, in certain embodiments, treating SLE (e.g., lupus nephritis) in a subject using a method of the disclosure results in a decrease in disease activity as measured by an SLE disease activity index or scale, such as the SLEDAI-2K responder index, the British Isles Lupus Assessment Group (BILAG) index, the Physician Global Assessment scale, or the Cutaneous LE Disease Area and Severity Index (CLASI). In certain embodiments following treatment of the subject for SLE, the subject meets the complete renal remission (CRR) criteria, the SRI-4 responder criteria, the SRI- 5 responder criteria, the SRI-6 responder criteria, the BICLA responder criteria, the LLDAS criteria, the DORIS remission criteria, and / or the count of tender and swollen joints.

[0142] In certain embodiments wherein a therapeutic method of the disclosure is used to treat SLE in a subject in need thereof, the subject’s SLEDAI-2K score is decreased. For example, in certain embodiments, the subject’s SLEDAI-2K score is decreased by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, or at least 30, e.g., as compared to the subject’s score prior to administration of the treatment, or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, the subject’s SLEDAI-2K score is reduced to 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2, or 0-1. In certain embodiments, the subject’s SLEDAI-2K score is reduced to 5, 4, 3, 2, 1, or 0. In certain embodiments, the subject’s SLEDAI-2K score is reduced to 0.

[0143] In certain embodiments wherein a therapeutic method of the disclosure is used to treat SLE in a subject in need thereof, the subject’s BILAG-2004 score decreases. For example, in certain embodiments, the subject’s BILAG score decreases by at least 1 grade, by at least 2 grades, or by 3 grades, e.g., as compared to the subject’s BILAG score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, the subject’s BILAG score decreases by 1, 2, or 3 grades. In certain embodiments, the subject’s BILAG score decreases to grade B, to grade C, or to grade D.

[0144] In certain embodiments wherein a therapeutic method of the disclosure is used to treat SLE in a subject in need thereof, the subject’s PGA score decreases after being treated inaccordance with a method of disclosure. For example, in certain embodiments, the subject’s PGA score decreases by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.8, at least 1, at least 1.5, at least 2, or at least 2.5 e.g., as compared to the subject’s PGA score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment.

[0145] In certain embodiments wherein a therapeutic method of the disclosure is used to treat SLE in a subject in need thereof, the subject’s CLASI score decreases after being treated in accordance with a method of disclosure. For example, in certain embodiments, the subject’s CLASI score decreases by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, or at least 30, e.g., as compared to the subject’s CLASI score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment.

[0146] In certain embodiments, therapeutic methods of the disclosure may be used to treat myositis (e.g., juvenile myositis) in a subject. In certain embodiments, treating myositis in a subject using a method of the disclosure results in a decrease in a disease symptom or activity as measured by an appropriate index, assessment, or scale, e.g., any of the following: MMT-8, Patient Global Assessment of Disease Activity (PGA), Physician Global Assessment of Disease Activity (MDGA), Health Assessment Questionnaire Disability Index (HAQ-DI), Extramuscular Global Assessment (Myositis Disease Activity Assessment Tool (MDAAT)), and Pain and Fatigue Numeric Rating Scale (NRS).

[0147] In certain embodiments, treating myositis (e.g., juvenile myositis) in a subject using a method of the disclosure results in a decrease in the serum level of an enzyme in the subject, e.g., creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), aldolase, myoglobin, or lactate dehydrogenase (LDH). For example, in certain embodiments, the serum level of the enzyme in the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, e.g., as compared to the serum level of the enzyme in the subject prior toadministering the treatment, or as compared to an appropriate control subject or group that did not receive the treatment.

[0148] In certain embodiments, therapeutic methods of the disclosure may be used to treat pemphigus vulgaris in a subject. In certain embodiments, treating pemphigus vulgaris in a subject using a method of the disclosure results in a decrease in a disease symptom or disease activity as measured by an appropriate index, assessment, or scale, e.g., the Pemphigus Disease Activity Index (PDAI). For example, in certain embodiments, the PDAI score of the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, e.g., as compared to the score of the subject prior to administering the treatment, or as compared to an appropriate control subject or group that did not receive the treatment.

[0149] In certain embodiments, therapeutic methods of the disclosure may be used to treat myasthenia gravis (e.g., MuSK myasthenia gravis) in a subject. In certain embodiments, treating myasthenia gravis in a subject using a method of the disclosure results in a decrease in a disease symptom or activity as measured by an appropriate index, assessment, or scale, e.g., any of the following: the Myasthenia Gravis Composite scale (MG Composite), the Myasthenia Gravis activities of daily living profile (MG-ADL), the improved 15-item myasthenia gravis quality of life scale (MG-QOL 15r), and / or the quantitative Myasthenia Gravis scoring system (QMG). For example, in certain embodiments, the subject’s MG Composite score decreases by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40, e.g, as compared to the subject’s MG Composite score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, the subject’s MG-ADL score decreases by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20, e.g, as compared to the subject’s MG-ADL score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, the subject’s MG-QOL 15r score decreases by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50, e.g., as compared to the subject’s MG-QOL 15r score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment. In certain embodiments, the subject’s QMG score decreases by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, or at least 30, e.g., as compared to the subject’s QMG score prior to being treated according to a method of the disclosure, or as compared to an appropriate control subject or group that did not receive the treatment.

[0150] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. However, it is understood that preconditioning agents are not included. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0151] In certain embodiments, a method or composition described herein is administered in combination with one or more additional therapies. In certain embodiments, the additional therapy may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferativecompound, e.g., a steroid, a biologic immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a bronchodilator, a statin, an anti-inflammatory agent (e.g. methotrexate), or an NSAID. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes. In certain embodiments, the additional therapy may be an antibody therapy (e.g., belimumab) or a corticosteroid.

[0152] The disclosure also provides compositions for use in any of the therapeutic methods disclosed herein. The composition may comprise, consistent essentially of, or consist of a dose of genetically modified immune cells as disclosed herein (e.g., a CAR immune cell as disclosed herein).III. Chimeric Antigen Receptors

[0153] Exemplary CARs for use in the therapeutic methods of the disclosure are described in greater detail below, in addition to nucleic acids encoding such CARs, vectors comprising said nucleic acids, genetically engineered cells modified to express said CARs, and other related methods and compositions.

[0154] In certain embodiments, a CAR of the disclosure comprises (1) an extracellular binding domain (e.g., comprising an antigen-binding site) that provides specificity for a desired target (e.g., a protein or antigen, such as an immune cell surface antigen), (2) a transmembrane domain, (3) an intracellular signaling domain and, optionally, (4) a costimulatory domain. In certain embodiments, the intracellular signaling domain is, or is derived from, a stimulatory molecule, such as a T cell activating domain providing a primary activation signal. Upon specific binding to the target , the receptor generally delivers an immunostimulatory signal, such as an IT AM- transduced signal, into the cell, thereby activating the cell and promoting a targeted immune response. In certain embodiments, the CAR further comprises one or more costimulatory signaling domains comprising functional signaling domains derived from one or more costimulatory molecules. In certain embodiments, a CAR of the disclosure comprises a KIR transmembrane domain and a KIR cytoplasmic domain. Further examples of CARs are provided in U.S. Patent Nos. 7,446,190 and 9,181,527, U.S. Patent Application Publication Nos. 2016 / 0340406 and 2017 / 0049819, and International Patent Application Publication No.WO20 18 / 140725.a. CAR Extracellular Domain

[0155] The extracellular domain of the CAR comprises a binding domain that binds a surface protein of a target cell, e.g., a B cell. The extracellular binding domain can comprise, for example, an antigen-binding site that specifically binds an antigen on a target cell (e.g., a B-cell). Alternatively, the extracellular domain can comprise, for example, an autoantigen that specifically binds to autoantibodies, e.g, an autoantibody on the surface of an autoantibodyproducing B cell (in the form of a BCR). i. Antigen-binding Sites

[0156] In certain embodiments, the extracellular domain of the CAR comprises an antigenbinding site that specifically binds a target antigen. For example, in certain embodiments, the extracellular antigen-binding domain comprises an antigen-binding fragment of an antibody or a derivative thereof. In certain embodiments, the extracellular domain comprises a Fab fragment or an scFv. In certain embodiments, the extracellular domain comprises an scFv. In certain embodiments, the antigen binding site is present in the Fab or the scFv.

[0157] In certain embodiments, the target antigen specifically bound by the antigen binding site is a polypeptide. In certain embodiments, the target antigen is selectively expressed or overexpressed on cells of a particular cell type. In certain embodiments, the targeted antigen is expressed by an immune cell, e.g, a B cell. In certain embodiments, the targeted antigen is present on the surface of an immune cell, e.g., a B cell. In certain embodiments, the antigen is a B cell marker. In certain embodiments, the antigen targeted by the CAR is CD 19, CD20, BCMA, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b or CD30. In certain embodiments, the antigen is CD 19.

[0158] As described herein, an antigen-binding site of a CAR of the disclosure may comprise: (a) an immunoglobulin heavy chain variable region (VH) comprising the structure CDRHI- CDRH2-CDRH3 and (b) an immunoglobulin light chain variable region (VL) comprising the structure CDRLI-CDRL2-CDRL3, wherein the VH and the VL together define a single binding site for binding a targeted antigen. In certain embodiments, the VH and VL each comprises one or more framework (FR) regions (e.g., 1, 2, 3, or 4 framework regions). In certain embodiments, the VH comprises the structure FRHI-CDRHI-FRH2-CDRH2-FRH3-CDRH3-FRH4 and / or the VL comprises the structure FRL1-CDRLI-FRL2-CDRL2-FRL3-CDRL-FRL4.

[0159] Exemplary antigen-binding sites that bind CD19 and that can be used in a CAR of the disclosure are described in greater detail hereinbelow.

[0160] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH that comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an antigen-binding site disclosed in TABLE 1, 2, or 3, and a VL that comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same antigen-binding site disclosed in TABLE 1, 2, or 3. In certain embodiments, the antigen-binding site that binds CD 19 comprises CDRHI, CDRH2, CDRHS, CDRLI, CDRL2, and CDRLS sequences present in the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8, which can be identified using CDR determination algorithms known in the art, for example, the algorithms disclosed herein. In certain embodiments, the antigen-binding site that binds CD 19 comprises CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences present in the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 17, which can be identified using CDR determination algorithms known in the art, for example, the algorithms disclosed herein. In certain embodiments, the antigen-binding site that binds CD19 comprises CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences present in the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO: 48, which can be identified using CDR determination algorithms known in the art, for example, the algorithms disclosed herein. In certain embodiments, the antigen-binding site comprises CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences determined under IMGT (see Lefranc, (1999) THE IMMUNOLOGIST, 7, 132-136), e.g., as indicated in TABLE 1, where the CDR sequences in each of the VH and VL sequences are underlined and then denoted individually. In certain embodiments, the antigen-binding site comprises CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences determined under Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), e.g., as indicated in TABLE 2, where the CDR sequences in each of the VH and VL sequences are underlined and then denoted individually. In certain embodiments, the antigen-binding site comprises CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences determined under Kabat (see Kabat el al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), e.g.,as indicated in TABLE 3, where the CDR sequences in each of the VH and VL sequences are underlined and then denoted individually. In certain embodiments, the antigen-binding site comprises CDRHI, CDRH2, CDRHS, CDRLI, CDRL2, and CDRLS sequences determined under MacCallum (see, MacCallum R M et al. , (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art, of the VH and VL sequences of an antibody disclosed in TABLE 1, 2, or 3. Identification of CDR and framework sequences is within the level of ordinary skill in the art, and it is understood that the boundaries between CDR and framework sequences may depend upon the definition or convention that is used (e.g., IMGT, Kabat, Chothia, etc.).

[0161] In certain embodiments, the antigen-binding site comprises the CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences of an antigen-binding site disclosed in TABLE 1, where the CDR sequences in each of the VH and VL sequences are underlined and defined by the IMGT convention. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antigen-binding site disclosed in TABLE 1.TABLE 1. Exemplary anti-CD19 antigen-binding sites (IMGT)

[0162] In certain embodiments, the antigen-binding site comprises the CDRHI, CDRH2,CDRHS, CDRLI, CDRL2, and CDRLS sequences of an antigen-binding site disclosed in TABLE 2, where the CDR sequences in each of the VH and VL sequences are underlined and defined by the Chothia convention. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antigen-binding site disclosed in TABLE 2.TABLE 2. Exemplary anti-CD19 antigen-binding sites (Chothia)

[0163] In certain embodiments, the antigen-binding site comprises the CDRHI, CDRH2,CDRHS, CDRLI, CDRL2, and CDRLS sequences of an antigen-binding site disclosed in TABLE 3, where the CDR sequences in each of the VH and VL sequences are underlined and defined by the Kabat convention. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antigen-binding site disclosed in TABLE 3.TABLE 3. Exemplary anti-CD19 antigen-binding sites (Kabat)

[0164] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRm, CDRH2, and CDRHS sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, wherein CDRHI, CDRH2, and CDRHS sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRHS sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0165] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRH2, and CDRHS sequences set forth as SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, wherein CDRHI, CDRH2, and CDRHS sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRHS sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.1

[0166] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRm, CDRH2, and CDRHS sequences set forth as SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively, wherein CDRm, CDRH2, and CDRHS sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NO: 106, GAS, and SEQ ID NO: 47, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRHS sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0167] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRm, CDRm, and CDRHS sequences set forth as SEQ ID NOs: 30, 31, and 32, respectively, wherein CDRm, CDRm, and CDRm sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NOs: 33, 34, and 35, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRm, CDRm, and CDRm sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0168] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRm, CDRm, and CDRm sequences set forth as SEQ ID NOs: 36, 37, and 38, respectively, wherein CDRm, CDRm, and CDRm sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NOs: 39, 40, and 16, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRm, CDRm, and CDRm sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0169] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRm, CDRm, and CDRm sequences set forth as SEQ ID NOs: 108, 109, and 43, respectively, wherein CDRm, CDRm, and CDRm sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NOs: 45, 46, and 47, respectively, wherein CDRLI, CDRL2, and CDRL3sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRHS sequences and / or the CDRLI, CDRL2, and CDRLS sequences are interposed between human or humanized immunoglobulin FR sequences.

[0170] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth as SEQ ID NOs: 110, 111, and 32, respectively, wherein CDRHI, CDRH2, and CDRH3 sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NOs: 33, 34, and 35, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRH3 sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0171] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth as SEQ ID NOs: 112, 113, and 38, respectively, wherein CDRHI, CDRH2, and CDRH3 sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NOs: 39, 40, and 16, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRH3 sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0172] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth as SEQ ID NOs: 41, 42, and 43, respectively, wherein CDRHI, CDRH2, and CDRH3 sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth as SEQ ID NOs: 45, 46, and 47, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certain embodiments, the CDRHI, CDRH2, and CDRH3 sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0173] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising the amino acid sequence of SEQ ID NO: 4, 13, or 44. In certain embodiments, theantigen-binding site that binds CD 19 comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, 17, or 48.

[0174] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising the amino acid sequence of SEQ ID NO: 4, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0175] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising the amino acid sequence of SEQ ID NO: 13, and / or a VL comprising the amino acid sequence of SEQ ID NO: 17.

[0176] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising the amino acid sequence of SEQ ID NO: 44, and / or a VL comprising the amino acid sequence of SEQ ID NO: 48.

[0177] In certain embodiments, an antigen-binding site that binds CD 19 comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from any one of SEQ ID NOs: 4, 13, and 44. Alternatively or in addition, an antigen-binding site that binds CD19 comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from any one of SEQ ID NOs: 8, 17, and 48.

[0178] In certain embodiments, an antigen-binding site that binds CD 19 comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 4; and / or comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%,at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 8.

[0179] In certain embodiments, an antigen-binding site that binds CD 19 comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected of SEQ ID NO: 13; and / or comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 17.

[0180] In certain embodiments, an antigen-binding site that binds CD 19 comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected of SEQ ID NO: 44; and / or comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 48.

[0181] In each of the foregoing embodiments, it is contemplated herein that VH sequences and / or VL sequences that together bind CD 19 may each independently contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the VH and / or the VL. In certain embodiments, a VH sequence and / or a VL sequence that together bind CD19 may each independently contain 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2- 5, 3-4, 3-5, or 4-5 amino acid alterations (e.g., substitutions, deletions, or additions) in the framework regions of the VH and / or the VL. In certain embodiments, the framework regions ofthe VH are humanized or human framework regions. In certain embodiments, the framework regions of the VL are humanized or human framework regions.

[0182] In certain embodiments, an antigen-binding site that binds CD 19 is present in an scFv. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 51.

[0183] In certain embodiments, an antigen-binding site that binds CD 19 is present in an scFv, wherein the scFv comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 9.

[0184] In certain embodiments, an antigen-binding site that binds CD 19 is present in an scFv, wherein the scFv comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected of SEQ ID NO: 18.

[0185] In certain embodiments, an antigen-binding site that binds CD 19 is present in an scFv, wherein the scFv comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected of SEQ ID NO: 51.

[0186] In certain embodiments, an antigen-binding site that binds CD 19 is humanized or fully human. In certain embodiments, the antigen-binding site that binds CD 19 is fully human.

[0187] Further examples of antigen-binding sites that bind CD19 are provided in U.S. Patent Nos. 7,446,179, 9,765,156, 10,125,193, 10,221,245, 10,287,350, 10,301,388, 10,457,730, 10,493,139, 10,533,055, 10,662,248, 10,780,118, 10,844,120, 10,874,693, 11,001,639,11,034,750, 11,034,763, 11,077,144, and 11,141,436; U.S. Patent Application Publication Nos. 2020 / 0038443, 2020 / 0062843, 2020 / 0123254, 2020 / 0289563, 2020 / 0376033, 2020 / 0384023, 2020 / 0384026, 2021 / 0002366, 2021 / 0061907, 2021 / 0069244, 2021 / 0101978, 2021 / 0196756, 2021 / 0238253, 2021 / 0332133, 2021 / 0395362; and 2021 / 0395364, and International Patent Application Publication Nos. 2018 / 201794, 2019 / 137518, 2019 / 154313, 2019 / 214332, 2020 / 233589, 2021 / 170146, 2021 / 217130, 2021 / 223719, 2021 / 223720, 2021 / 225532, and 2022 / 012683.

[0188] In certain embodiments, the antigen-binding site binds CD 19 with a KD of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or stronger, as measured using standard binding assays, for example, surface plasmon resonance or bio-layer interferometry. In certain embodiments, the antigenbinding site binds CD 19 with a KD of from about 20 nM to about 0.05 nM, from about 20 nM to about 0.075 nM, from about 20 nM to about 0.1 nM, from about 20 nM to about 0.5 nM, from about 20 nM to about 1 nM, from about 10 nM to about 0.05 nM, from about 10 nM to about 0.075 nM, from about 10 nM to about 0.1 nM, from about 10 nM to about 0.5 nM, from about 10 nM to about 1 nM, from about 5 nM to about 0.05 nM, from about 5 nM to about 0.075 nM, from about 5 nM to about 0.1 nM, from about 5 nM to about 0.5 nM, from about 5 nM to about 1 nM, from about 3 nM to about 0.05 nM, from about 3 nM to about 0.075 nM, from about 3 nM to about 0.1 nM, from about 3 nM to about 0.5 nM, from about 3 nM to about 1 nM, from about 3 nM to about 2 nM, from about 2 nM to about 0.05 nM, from about 2 nM to about 0.075 nM, from about 2 nM to about 0.1 nM, from about 2 nM to about 0.5 nM, from about 2 nM to about 1 nM, from about 1 nM to about 0.05 nM, from about 1 nM to about 0.075 nM, from about 1 nM to about 0.1 nM, from about 1 nM to about 0.5 nM, from about 0.5 nM to about 0.05 nM, from about 0.5 nM to about 0.075 nM, from about 0.5 nM to about 0.1 nM, from about 0.1 nM to about 0.05 nM, from about 0.1 nM to about 0.075 nM, or from about 0.075 nM to about 0.05 nM, or from about 0.05 nM to about 0.035 nM, as measured using standard binding assays, for example, surface plasmon resonance or bio-layer interferometry.

[0189] In certain embodiments, the antigen-binding site that binds CD 19 cross-competes with an antigen-binding site disclosed in TABLE 1, 2, or 3. Competition assays for determining whether an antigen-binding site binds to the same epitope as, or competes for binding with a disclosed antibody are known in the art. Exemplary competition assays include immunoassays(e.g., ELISA assays, RIA assays), surface plasmon resonance, (e.g., BIAcore analysis), bio-layer interferometry, and flow cytometry.

[0190] The antigen-binding sites disclosed herein may be further optimized (e.g., affinity- matured) to improve biochemical characteristics including affinity and / or specificity, improve biophysical properties including aggregation, stability, precipitation and / or non-specific interactions, and / or to reduce immunogenicity. Affinity-maturation procedures are within ordinary skill in the art. For example, diversity can be introduced into an immunoglobulin heavy chain and / or an immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-specific mutagenesis.

[0191] Generally, an optimized antigen-binding site has at least the same, or substantially the same, affinity for the antigen as the non-optimized (or parental) antigen-binding site from which it was derived. Preferably, an optimized antibody has a higher affinity for the antigen when compared to the parental antibody.

[0192] The functional ability of a CAR to specifically bind to its target antigen (e.g., CD 19) can be assessed in a Jurkat reporter cell line, wherein activation of the CAR is dependent on binding to plate-bound or cell-bound target protein (in response to which the activated cells fluoresce green due to an NFAT-GFP reporter construct contained therein). Such methods are useful and reliable qualitative measures for functional binding ability.

[0193] In certain embodiments, the extracellular binding domain of the CAR comprises means for binding CD 19. In certain embodiments, the means is an antigen-binding site, e.g, an antigen-binding site that binds CD 19 described herein. ii. Autoantigens

[0194] In certain embodiments, the extracellular binding domain of the CAR comprises an autoantigen. The autoantigen can bind to autoantibodies, such as autoantibodies on the surface of a BCR-expressing B-cell. The particular autoantigen used in the CAR may be determined based on the autoimmune disease to be treated, wherein the autoantigen is derived from a protein (or fragment thereof) to which autoantibodies associated with the disease specifically bind. For example, for treating MuSK-associated myasthenia gravis (an autoimmune disease involving B- cells that produce anti-MuSK autoantibodies) the extracellular binding domain of the CAR maycomprise a MuSK autoantigen, enabling the CAR to specifically bind to an anti-MuSK autoantibody (BCR) on the surface of a B cell. Similarly, for AChR-associated myasthenia gravis, the CAR can comprise an AChR autoantigen, enabling the CAR to specifically bind to an anti-AChR autoantibody (BCR) on the surface of a B cell. In certain embodiments, the autoantigen is selected from a Dsgl autoantigen, a Dsg3 autoantigen, a MuSK autoantigen, an AChR autoantigen, and a PLA2R autoantigen. In certain embodiments, the autoantigen is not a Dsg autoantigen. In certain embodiments, the autoantigen not a Dsg3 autoantigen and / or not a human Dsgl autoantigen.

[0195] In certain embodiments, the autoimmune disease to be treated with the CAR is MuSK- associated myasthenia gravis, and the extracellular binding domain of the CAR comprises a MuSK autoantigen. In certain embodiments, the autoimmune disease to be treated with the CAR is AChR-associated myasthenia gravis, and the extracellular binding domain of the CAR comprises an AChR autoantigen. In certain embodiments, the autoimmune disease to be treated is glomerular disease or primary membranous nephropathy, and the extracellular binding domain of the CAR comprises a PLA2R autoantigen. In certain embodiments, the autoimmune disease to be treated is pemphigus vulgaris, and the extracellular binding domain of the CAR comprises a Dsg3 autoantigen and / or a Dsgl autoantigen.

[0196] In certain embodiments, it is beneficial for the autoantigen to be derived from a protein of the same species to be treated. For example, for use in humans, it may be beneficial for the CAR autoantigen to be derived from a human protein that specifically binds human autoantibodies. For example, in certain embodiments, the autoantigen is selected from a human Dsgl autoantigen, a human Dsg3 autoantigen, a human MuSK autoantigen, a human AChR autoantigen, and a human PLA2R autoantigen. In certain embodiments, the autoantigen is not a human Dsg autoantigen. In certain embodiments, the autoantigen not a human Dsg3 autoantigen and / or not a human Dsgl autoantigen.

[0197] In certain embodiments, the CAR extracellular binding domain comprises a MuSK autoantigen. In certain embodiments, the MuSK autoantigen comprises the full-length extracellular domain of wild-type MuSK (e.g., as in SEQ ID NO: 54). In certain embodiments, the MuSK autoantigen has a substitution of an isoleucine at a position corresponding to position 96 of wild-type human MuSK. In certain embodiments, the isoleucine at a positioncorresponding to position 96 of wild-type human MuSK is substituted with alanine (e.g., as in SEQ ID NO: 55).

[0198] In certain embodiments, the CAR extracellular binding domain comprises a MuSK autoantigen, and the MuSK autoantigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54 and 55. In certain embodiments, the MuSK autoantigen comprises an amino acid sequence at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 54 and 55. In certain embodiments, the MuSK autoantigen comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0199] In certain embodiments, the CAR extracellular binding domain comprises a MuSK autoantigen, and the MuSK autoantigen comprises a conservative substitution relative to the amino acid sequence of SEQ ID NO: 54 or 55. In certain embodiments, the MuSK autoantigen comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55. In certain embodiments, the MuSK autoantigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0200] In certain embodiments, the CAR extracellular binding domain comprises an AChR autoantigen, and the AChR autoantigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-67. In certain embodiments, the AChR autoantigen comprises an amino acid sequence at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-67. In certainembodiments, the AChR autoantigen comprises the amino acid sequence of any one of SEQ ID NOs: 60-67.

[0201] In certain embodiments, the CAR extracellular binding domain comprises an AChR autoantigen, and the AChR autoantigen comprises a conservative substitution relative to the amino acid sequence of any one of SEQ ID NOs: 60-67. In certain embodiments, the AChR autoantigen comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., the amino acid sequence of any one of SEQ ID NOs: 60-67. In certain embodiments, the AChR autoantigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more conservative substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 60-67.

[0202] In certain embodiments, the CAR extracellular binding domain comprises a PLA2R autoantigen, and the PLA2R autoantigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-82 and 93-95. In certain embodiments, the PLA2R autoantigen comprises an amino acid sequence at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-82 and 93-95. In certain embodiments, the PLA2R autoantigen comprises the amino acid sequence of any one of SEQ ID NOs: 75-82 and 93-95.

[0203] In certain embodiments, the CAR extracellular binding domain comprises a PLA2R autoantigen, and the PLA2R autoantigen comprises a conservative substitution relative to the amino acid sequence of any one of SEQ ID NOs: 75-82 and 93-95. In certain embodiments, the PLA2R autoantigen comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g, the amino acid sequence of any one of SEQ ID NOs: 75-82 and 93-95. In certain embodiments, the PLA2R autoantigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more conservative substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 75-82 and 93-95.

[0204] In certain embodiments, the CAR extracellular binding domain comprises a Dsg3 autoantigen, and the Dsg3 autoantigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99 and 100. In certain embodiments, the Dsg3 autoantigen comprises an amino acid sequence at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 99 and 100. In certain embodiments, the Dsg3 autoantigen comprises the amino acid sequence of any one of SEQ ID NOs: 99 and 100.

[0205] In certain embodiments, the CAR extracellular binding domain comprises a Dsg3 autoantigen, and the Dsg3 autoantigen comprises a conservative substitution relative to the amino acid sequence of any one of SEQ ID NOs: 99 and 100. In certain embodiments, the Dsg3 autoantigen comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., the amino acid sequence of any one of SEQ ID NOs: 99 and 100. In certain embodiments, the Dsg3 autoantigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more conservative substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 99 and 100. b. CAR Transmembrane Domain

[0206] The extracellular antigen-binding domain can be fused to the transmembrane domain of the CAR. In certain embodiments, the transmembrane domain of the CAR is derived from a naturally occurring transmembrane protein. In certain embodiments, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. In certain embodiments, the transmembrane domain comprises the transmembrane region(s) of one or more proteins selected from the group consisting of TCR a chain, TCR 0 chain, TCR chain, CD28, CD3s, CD45, CD4, CD5, CD8a, CD9, CD 16, CD22, EGFR, CD37, CD64, CD80, CD86,CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R0, IL2Ry, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA- 1, ITGAM, CD1 lb, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, and NKG2C. In certain embodiments, the transmembrane comprises the transmembrane region(s) of one or more proteins selected from the group consisting of CD8a, CD28, CD3^, and CD4. In certain embodiments, the transmembrane comprises the transmembrane region(s) of one or more proteins selected from the group consisting of CD8a and CD28. In certain embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In certain embodiments, the transmembrane domain is one that naturally is associated with one of the domains (e.g., primary signaling domain or co-stimulatory signaling domain) in the CAR. In certain embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid multimerization with a transmembrane domain of the same or a different surface membrane protein, thereby to minimize interactions with other members of a receptor complex (e.g., the CAR complex). In other embodiments, the transmembrane domain is capable of homodimerization with another CAR on the immune cell (e.g., T cell) surface. In certain embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same immune cell.

[0207] In certain embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In certain embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19.

[0208] In certain embodiments, the transmembrane domain comprises a conservative substitution relative to a transmembrane domain disclosed herein, e.g., relative to a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, or less than 15 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 19.

[0209] In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In certain embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52.

[0210] In certain embodiments, the transmembrane domain comprises a conservative substitution relative to a transmembrane domain disclosed herein, e.g., relative to a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the transmembrane domain comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, or less than 15 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the transmembrane domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 52.

[0211] In certain embodiments, the CAR comprises a transmembrane domain and / or a cytoplasmic (intracellular) domain from a killer immunoglobulin-like receptor (KIR) family protein. The KIR gene family has at least 15 gene loci (KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 KIR3DL1 / S1 KIR3DL2 KIR3DL3) and two pseudogenes (KIR2DP1 and KIR3DP1) encoded within a 100-200 Kb region of the Leukocyte Receptor Complex (LRC) located on chromosome 19 (19ql3.4). The LRC constitutes a large, 1 Mb, and dense cluster of rapidly evolving immune genes which contains genes encoding other cell surface molecules with distinctive Ig-like extra- cellular domains. In addition, the extended LRC contains genes encoding the transmembrane adaptor molecules DAP 10 and DAP 12. Thus, in certain embodiments, a cell comprising the CAR of the disclosure comprising a KIR transmembrane domain and / or cytoplasmic domain may also comprise a polynucleotide encoding DAP 10 or DAP 12. In certain embodiments, the KIR is KIRS2 or KIR2DS2. c. CAR Intracellular Domains

[0212] The intracellular domain of the CAR comprises an intracellular signaling domain (i.e., a functional signaling domain derived from a stimulatory molecule) and, optionally, one or more costimulatory signaling domains (i.e., functional signaling domains derived from at least one costimulatory molecule). These intracellular signaling and costimulatory domains are responsible, at least in part, for an immune cell response, including, but not limited to, proliferation, differentiation, and activation of a specialized function of the immune cell (e.g., cytotoxic activity or secretion of cytokines of a T cell) in which the CAR is expressed. The intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0213] Intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of IT AM- containing cytoplasmic signaling sequences that are of particular use in the CARs of the present application include those derived from CD3( / , common FcRy (FCER1G), FcyRIIa, FcR0 (FceRlb), CD3y, CD38, CD3s, CD79a, CD79b, DAP10, and DAP12. In certain embodiments, the intracellular signaling domain of a CAR described herein comprises a functional, cytoplasmic signaling domain derived from CD3( / , FcRy, FcR0, CD3y, CD38, CD3s, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, common FcRy (FCER1G), FcyRIIa, FcR0 (FcsRlb), DAP10, and / or DAP12. In certain embodiments, the intracellular signaling domain comprises a CD3( / signaling domain or an FcRy signaling domain.

[0214] In certain embodiments, a CAR of the disclosure comprises a CD3( / signaling domain by itself or in combination with any other desired cytoplasmic domain(s) useful in the context ofthe CAR. For example, the CAR can comprise a CD3^ chain portion (i.e., a CD3^ intracellular signaling domain) and an intracellular domain of a costimulatory molecule, for example, a 4- 1BB intracellular domain or a CD28 intracellular domain. In certain embodiments, a CAR of the disclosure comprises a CD3^ signaling domain and a 4- IBB costimulatory domain. In certain embodiments, a CAR of the disclosure comprises a CD3^ signaling domain and a CD28 costimulatory domain. In certain embodiments, the CD3^ intracellular signaling domain is a human T-cell surface glycoprotein CD3^ chain isoform 3 (human CD247) intracellular domain. The human CD3^ intracellular domain provides stimulatory intracellular signaling upon binding of the extracellular antigen to its ligand without HLA restriction.

[0215] In certain embodiments, a CAR of the disclosure comprises a CD3^ signaling domain. In certain embodiments, the intracellular signaling domain comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 21.

[0216] In certain embodiments, the intracellular signaling domain comprises a conservative substitution relative to an intracellular signaling domain disclosed herein, e.g., relative to an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, or less than 15 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 21.

[0217] In certain embodiments, a CAR of the disclosure comprises a costimulatory domain. A costimulatory domain comprises a functional signaling domain derived from a costimulatory molecule, a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of costimulatory molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1, CDl la / CD18), CD2, CD7, CD258 (LIGHT), NKG2C, B7-H3, CD83 ligands, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD80, IL2R0, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, and PAG / Cbp. In certain embodiments, a costimulatory domain of the CAR comprises a functional signaling domain of a costimulatory molecule described herein, e.g., 0X40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a CD83 ligand, ICAM-1, LFA-1 (CD1 la / CD18), ICOS and 4-1BB (CD137), or any combination thereof. In certain embodiments, a costimulatory domain of the CAR comprises a functional signaling domain of a costimulatory molecule selected from 4-1BB (CD137), CD28, ICOS, CD27, CD40, and 0X40. In certain embodiments, the CAR comprises a costimulatory domain comprising a 4-1BB intracellular domain and / or a CD28 intracellular domain.

[0218] In certain embodiments, a CAR of the disclosure comprises a costimulatory domain comprising a 4- IBB intracellular domain. In certain embodiments, the costimulatory domain comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 20.

[0219] In certain embodiments, the costimulatory domain comprises a conservative substitution relative to a costimulatory domain disclosed herein, e.g., relative to costimulatory domain comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the costimulatory domain comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, or less than 15 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 20.

[0220] In certain embodiments, a CAR of the disclosure comprises a costimulatory domain comprising a CD28 intracellular domain. In certain embodiments, the costimulatory domain comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 53.

[0221] In certain embodiments, the costimulatory domain comprises a conservative substitution relative to a costimulatory domain disclosed herein, e.g., relative to costimulatory domain comprising the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the costimulatory domain comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, or less than 15 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the intracellular signaling domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 53.

[0222] The intracellular signaling and costimulatory domains within the cytoplasmic portion of the CAR may be linked to each other in a random or specified order. In certain embodiments, a costimulatory signaling domain is deployed N-terminal to the primary signaling domain. Optionally, the intracellular domains are linked by a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids in length. In certain embodiments, the intracellular domains are linked by, e.g., a GS doublet or by a (G4S)nlinker. In certain embodiments, the intracellular signaling domain (e.g. CD3^ signaling domain) is the C-terminal domain of the CAR. d. Other CAR Domains

[0223] The extracellular antigen-binding domain of the CAR can be connected to the transmembrane domain by a hinge domain or linker. In certain embodiments, the hinge domain or linker is interposed between the antigen binding site and the transmembrane domain. Avariety of hinges or linkers can be employed, including, but not limited to, the human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a Gly-Ser linker, a (GIS)4 linker, a KIR2DS2 hinge, and a CD8a hinge.

[0224] In certain embodiments, the hinge domain is a CD8a hinge. In certain embodiments, the hinge domain comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 22.

[0225] In certain embodiments, the hinge domain comprises a conservative substitution relative to a hinge domain disclosed herein, e.g., relative to a hinge domain comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the hinge domain comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, or less than 15 conservative substitutions relative to, e.g., the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the hinge domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 22.

[0226] In certain embodiments, the CAR comprises a CD8a transmembrane domain and a CD8a hinge.

[0227] In certain embodiments, the CAR comprises a signal peptide. In certain embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence encoding a signal peptide. In certain embodiments, the signal peptide is derived from a native polypeptide. In other embodiments, the signal peptide comprises a heterologous or non-native signal peptide. In certain embodiments, the signal peptide is a CD8a signal peptide or an IgG signal peptide.

[0228] In certain embodiments, the CAR comprises a CD8a signal peptide. In certain embodiments, the signal peptide comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence ofSEQ ID NO: 25. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the CAR comprises an IgG signal peptide. In certain embodiments, the signal peptide comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 26.

[0229] In certain embodiments, the signal peptide comprises a conservative substitution relative to a signal peptide disclosed herein, e.g., relative to a signal peptide comprising the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments, the signal peptide comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, or less than 10 conservative substitutions relative to, e.g., the signal peptide of SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments, the signal peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26. e. Exemplary CAR Constructs

[0230] In certain embodiments, a CAR of the disclosure comprises, in the N- to C-terminal direction: (1) an extracellular domain comprising an antigen binding site, for example, an scFv such as an anti-CD19 scFv, e.g., an scFv comprising the amino acid sequence of SEQ ID NO: 9; (2) an optional hinge domain or linker, for example, a CD8a hinge, e.g., a hinge comprising the amino acid sequence of SEQ ID NO: 22; (3) a transmembrane domain, for example, a CD8a transmembrane domain, e.g., a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 19; (4) an optional costimulatory domain, for example, an intracellular domain of 4-1BB, e.g., a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 20; and (e) an intracellular signaling domain, for example, a CD3^ signaling domain, e.g., a signaling domain comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the CAR further comprises an N-terminal signal peptide, for example, a CD8 signal peptide, e.g., a signal peptide comprising the amino acid sequence of SEQ ID NO: 25.Certain exemplary anti-CD19 CAR constructs of the disclosure are depicted in FIGURES 1A- 1B. An exemplary MuSK-CAR construct of the disclosure is depicted in FIGURE 1C.

[0231] In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 23. In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 27.

[0232] In certain embodiments, a CAR of the disclosure comprises a conservative substitution relative to a CAR disclosed herein, e.g., relative to a CAR comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 27. In certain embodiments, the CAR comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., the CAR of SEQ ID NO: 23 or SEQ ID NO: 27. In certain embodiments, the CAR comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 27.

[0233] In certain embodiments, the CAR is encoded by a nucleic acid at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29. In certain embodiments, the CAR is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0234] In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 49. In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 102.

[0235] In certain embodiments, a CAR of the disclosure comprises a conservative substitution relative to a CAR disclosed herein, e.g., relative to a CAR comprising the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 102. In certain embodiments, the CAR comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., the CAR of SEQ ID NO: 49 or SEQ ID NO: 102. In certain embodiments, the CAR comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or more conservative substitutions relative to the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 102.

[0236] In certain embodiments, the CAR is encoded by a nucleic acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 50. In certain embodiments, the CAR is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 50.

[0237] In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, 96-98, and 101. In certain embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, 96-98, and 101. In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 56-59, 68- 74, 83-92, and 96-98. In certain embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, and 96-98. In certain embodiments, a CAR of the disclosure comprises an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 56-59. In certain embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NOs: 56-59.

[0238] In certain embodiments, a CAR of the disclosure comprises a conservative substitution relative to a CAR disclosed herein, e.g., relative to a CAR comprising an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, 96-98, and 101. In certain embodiments, the CAR comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, 96-98, and 101. In certain embodiments, the CAR comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or more conservative substitutions relative to an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, 96-98, and 101. In certain embodiments, a CAR of the disclosure comprises a conservative substitution relative to a CAR disclosed herein, e.g., relative to a CAR comprising an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, and 96-98. In certain embodiments, the CAR comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutionsrelative to, e.g., an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, and 96-98. In certain embodiments, the CAR comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or more conservative substitutions relative to an amino acid sequence selected from SEQ ID NOs: 56-59, 68-74, 83-92, and 96-98. In certain embodiments, a CAR of the disclosure comprises a conservative substitution relative to a CAR disclosed herein, e.g., relative to a CAR comprising an amino acid sequence selected from SEQ ID NOs: 56-59. In certain embodiments, the CAR comprises less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 20, less than 25, or less than 30 conservative substitutions relative to, e.g., an amino acid sequence selected from SEQ ID NOs: 56-59. In certain embodiments, the CAR comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or more conservative substitutions relative to an amino acid sequence selected from SEQ ID NOs: 56-59. f. Vectors Comprising CAR-encoding Polynucleotides

[0239] In certain embodiments, CARs for use in the methods of the disclosure are encoded by polynucleotides which, optionally, may be present in a vector. In certain embodiments, the polynucleotide encodes a CAR comprising an extracellular domain, a transmembrane domain, an intracellular signaling domain and, optionally, a costimulatory domain. The extracellular domain comprises a binding domain which can comprise, e.g., an scFv (e.g., an anti-CD19 scFv) or an autoantigen (e.g., a MuSK autoantigen). In certain embodiments, the polynucleotide encodes a CAR comprising an anti-CD19 scFv, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3^ signaling domain.

[0240] In certain embodiments, the vector comprises a plasmid vector, viral vector, retroviral vector, lentiviral vector, adenoviral vector, AAV, retrotransposon (e.g., piggyback, sleeping beauty), site directed insertion vector (e.g., CRISPR, Zinc finger nucleases, TALEN), or suicide expression vector, or other known vector in the art.

[0241] In certain embodiments, the vector is a viral vector, e.g., a lentiviral vector, e.g., a 3rd generation lentiviral vector, e.g., a 3rdgeneration self-inactivating lentiviral vector.

[0242] Expression of the CAR can be verified by sequencing. Expression of the full length CAR protein may be verified using immunoblot, immunohistochemistry, flow cytometry, or other technology well known and available in the art.

[0243] The present disclosure also provides a vector in which DNA encoding the CAR of the present disclosure is inserted. Vectors, including those derived from retroviruses such as lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of resulting in low immunogenicity in the subject into which they are introduced.

[0244] In brief summary, the expression of natural or synthetic polynucleotides encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vector is one generally capable of replication in a mammalian cell, and / or also capable of integration into the cellular genome of the mammal. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0245] The nucleic acid can be cloned into any number of different types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0246] The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 2001 / 96584; WO 2001 / 29058; and U.S. Patent No. 6,326,193).

[0247] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0248] An example of a promoter that may be used is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the elongation factor-la promoter (EF-la), as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, or the hemoglobin promoter, as appropriate.

[0249] Further, the disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated for use in the methods and compositions of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence, which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. In certain embodiments, an inducible promoter is activated in response to an extracellular ligand. For example, in certain embodiments, the inducible promoter is activated (and the expression of the CAR is regulated) by an extracellular ligand binding to a synthetic receptor. For example, in certain embodiments, a synthetic receptor, e.g., a synthetic Notch receptor (i.e., “synNotch”) may be employed as a binding-triggered transcriptional switch that, when bound to its ligand, activates a promoter to which a nucleic acid sequence encoding the CAR is operably linked. Accordingly, as a nonlimiting example, such systems may require the presence of a ligand (e.g., to which the synNotch binds) for the immune cell to be responsive to a BCR or autoantibody (e.g., to which the CAR binds). The requirement of particular combinations to generate certain signaling outputs inmolecular circuits results in a logic gate. See, for example, Roybal et al., 2016 CELL 164(4):770- 9.

[0250] Examples of other systems for expressing or regulating expression of a chimeric receptor include those described in Wu etal. (2015) Science 350: aab4077; Fedorov etal. (2014) Cancer Journal 20:160-165; Kloss et al. (2013) Nature Biotechnology 31: 71-75; Sakemura et al. (2016) Cancer Immunol. Res. 4:658-668; Hill etal. (2018) NAT. CHEM. BIOL. 14: 112-117; Di Stasi et al. (2011) N. ENGL. J. MED. 365: 1673-1683; Budde et al. (2013) PLoS ONE 8: e82742; Wei etal. (2012) NATURE 488: 384-388; Ma etal. (2016) PROC. NATL. ACAD. SCI. USA 113: E450-458; Rodgers et al. (2016) PROC. NATL. ACAD. SCI. USA 113: E459-468; Kudo et al. (2014) CANCER RES. 74: 93-103, and Chen et al. (2010) PROC. NATL. ACAD. SCI. USA 107: 8531-8536.

[0251] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. The selectable marker may be carried on a separate piece of DNA or RNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0252] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA or RNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei etal., 2000 FEBS LETTERS 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may belinked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0253] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0254] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, micro injection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY).

[0255] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. RNA vectors include vectors having an RNA promoter and / other relevant domains for production of an RNA transcript. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0256] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of targeted delivery of nucleic acids are known, such as delivery of polynucleotides with targeted nanoparticles or another suitable submicron sized delivery system.

[0257] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome or lipid nanoparticle (LNP). The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of aliposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, lipid / RNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Examples of lipids, liposomes, lipid nanoparticles, and related formulations are described, for example, in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,006,417, 9,139,554, 9,364,435, 9,404,127, 9,415,109, 9,504,651, 9,518,272, 9,567,296, 9,580,711, 9,636,414, 9,694,077, 9,758,795, 9,814,777, 9,868,692, 9,878,042, 9,943,846, 9,950,065, 10,041,091, 10,106,490, 10,166,298, 10,221,127, 10,227,302, 10,233,148, 10,266,485, 10,442,756, 10,485,884, 10,561,732, 10,576,146, 10,577,403, 10,653,780, 10,703,789, 10,980,895, 11,045,418, 11,141,378, 11,173,120, 11,191,849, 11,285,222, 11,357,856, 11,446,383, 11,453,639, 11,478,552, 11,559,587, U.S. Patent Application Publication Nos. 2011 / 0117125, 2012 / 0264810, 2018 / 0000953, 2018 / 0085474, 2018 / 0185516, 2019 / 0022247, 2019 / 0032087, 2019 / 0274968, 2019 / 0336608, 2020 / 0046830, 2020 / 0109113, 2020 / 0155671, 2020 / 0163878, 2020 / 0164038, 2020 / 0172472, 2020 / 0297634, 2020 / 0297870, 2020 / 0306191, 2021 / 0145982, 2021 / 0207140, 2021 / 0220274, 2021 / 0346306, 2022 / 0000778, 2022 / 0001029, 2022 / 0118112, and 2022 / 0160899; and International Patent Application Publication Nos. 2008 / 042973, 2021 / 231929, and 2021 / 237084.

[0258] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C.Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh etal. (1991) GLYCOBIOLOGY 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0259] In certain embodiments wherein a non-viral delivery method is utilized, an exemplary delivery vehicle is a bioabsorbable silicon nanoparticle. Silicon nanoparticles can be made of either pure silicon, or a hydrolysable silicon-containing material, and can be made porous by standard techniques such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying a current. The silicon nanoparticles may be loaded with a polynucleotide, e.g., RNA, to be delivered into a host cell (e.g., in vitro, ex vivo, or in vivo). Silicon nanoparticles may be surface-treated with a lipid (e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), stearylamine (SA), and / or lecithin, which can aid in controlling the rate of release of the payload polynucleotide. A lipid-surface-treated silicon nanoparticle may be further treated with an amino acid (e.g., arginine, histidine, and / or glycine), which can promote stability of the payload nucleic acid, e.g, RNA. Examples of silicon nanoparticles are described, for example, in U.S. Patent No. 9,132,083, in U.S. Patent Application Publication Nos. 2022 / 0183989 and 2022 / 0184038, and in International Patent Application Publication No. 2020 / 193999.

[0260] Any domains and / or fragments of the CAR, vector, and the promoter may be synthesized gene fragments amplified by PCR or any other means known in the art.IV. Pharmaceutical Compositions

[0261] For therapeutic use, a genetically modified immune cell disclosed herein preferably is present in a pharmaceutical composition, optionally wherein the composition comprises a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition consists essentially of an effective amount of a genetically modified immune cell of the disclosure and a pharmaceutically acceptable carrier.

[0262] As described hereinabove, the engineered immune cells of the disclosure are useful in immune cell therapies (e.g., adoptive immune cell therapies, e.g, for the treatment of an autoimmune disease). Accordingly, the present disclosure provides compositions comprising one or more engineered immune cells as described herein. In certain embodiments, the one or more engineered immune cells are present in a pharmaceutical composition, e.g, wherein the composition comprises a pharmaceutically acceptable carrier.

[0263] In certain embodiments, the immune cell therapy is autologous, i.e., immune cells obtained from a patient, after in vitro culture, are administered to the same patient. In certain embodiments, the immune cell therapy is allogeneic and the immune cells are obtained from a healthy donor, optionally wherein the immune cells (e.g., T cells) are genetically engineered to inactivate or lack expression of a functional T cell receptor (TCR) and / or a human leukocyte antigen (HLA) molecule, e.g, an HLA class I component or an HLA class II component. For example, in certain embodiments, an allogeneic T cell can be engineered to have reduced or no expression of a functional TCR on its surface, or engineered to have reduced or no expression of one or more subunits that comprise a functional TCR (e.g, the TCR a chain or the TCR 0 chain). Alternatively, the T cell can express a functionally impaired TCR, e.g, by expression of mutated or truncated forms of one or more of the subunits of the TCR. In certain embodiments, an allogeneic T cell can be engineered to have reduced or no expression of a functional HLA molecule on its surface, e.g., reduced or no expression of an HLA class I molecule or an HLA class II molecule. In certain embodiments, surface expression of an HLA class I molecule is reduced in an allogeneic T cell by targeting or knocking out a sequence encoding beta-2 microglobulin (02M). In certain embodiments, surface expression of an HLA class II molecule is reduced in an allogeneic T cell by targeting or knocking out a sequence encoding CIITA. Such cells can be created through the use of a gene editing systems as described herein. Inembodiments, gene editing systems targeting sequences encoding TCR a chain, TCR 0 chain, 02 M, and / or CIITA are introduced into the cells, such that surface expression of functional TCR, HLA class I molecules, and / or HLA class II molecules is downregulated. In certain embodiments, the allogeneic T cell can lack a functional TCR and a functional HLA molecule, e.g., an HLA class I molecule and / or an HLA class II molecule..

[0264] An immune cell therapy can be provided as a cell composition. In certain embodiments, the engineered immune cell in the composition is an NK cell, e.g., a CARNK cell. In certain embodiments, the engineered immune cell in the composition is a T cell, e.g., a CAR T cell. It is understood that other types of cells, such as APCs, may be used for expanding T cells ex vivo. As such, the cell composition may include other cell types in addition to T cells. In certain embodiments, the cell composition has been enriched for T cells. Where the T cells are prepared by stimulation using APCs in an ex vivo cell culture, the T cells can be enriched by methods known in the art. For example, in certain embodiments, the APCs are removed from the cell culture by surface marker-based magnetic bead selection or cell sorting. In certain embodiments, the APCs are outgrown by T cells under conditions (e.g., cytokines) that preferably support T cell proliferation. In certain embodiments, the APCs are removed by their stronger adherence to tissue culture plate than T cells. The enrichment can produce a composition in which at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the composition are T cells. In certain embodiments, the composition is substantially free of myeloid cells. For example, in certain embodiments, the percentage of myeloid cells relative to all cells in the composition is 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0265] In certain embodiments, an immune cell composition may comprise one or more immunogenicity enhancing adjuvants (also referred to as “adjuvants” herein). Such adjuvants are substances that enhance or potentiate the immune response (e.g., immune responses mediated by CD8-positive T cells and helper-T (TH) cells to an antigen) in a non-antigen-specific manner, and would thus be considered useful in a pharmaceutical composition disclosed herein. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA®), resiquimod, IMUFACT®, IMP321, interleukins as IL-2, IL- 13, IL-21, interferon-a or -0, or pegylated derivatives thereof, IS Patch,ISS, ISCOMATRIX, ISCOMs, JUVIMMUNE®, LIPOVAC®, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP- EC, ONTAK®, OspA, poly(lactide coglycolide) [PLG]-based and dextran microparticles, talactoferrin SRL 172, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, QUIL®, or Superfos. Depending upon the circumstances, adjuvants such as Freund’s or GM-CSF may be preferred. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Ott etal. (1995) PHARM. BlOTECHNOL. 6: 277-96). In certain embodiments, an adjuvant is a naturally occurring adjuvant. In certain embodiments, an adjuvant is a non-naturally occurring adjuvant.

[0266] The immune cell therapy compositions can further comprise one or more carriers and / or excipients. Exemplary carriers and excipients are described herein (see the “Compositions Comprising Cyclophosphamide and / or Fludarabine” subsection above).

[0267] In certain embodiments of any of the therapeutic compositions or therapeutic methods described herein, a therapeutic agent (e.g., an engineered immune cell, such as a CAR-T cell) may be administered to a subject (e.g., a subject in need thereof) in a therapeutically effective amount. The therapeutically effective amount of the therapeutic agent (e.g., engineered immune cell) to be administered may depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the agent, the pharmaceutical formulation, and the route of administration. A preferred route of administration is intravenous infusion.

[0268] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components,or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist of the recited components, and that there are processes and methods according to the present invention that consist of the recited processing steps.

[0269] Throughout the description, where compositions are described as not including particular components, it should be understood that the compositions may also “not have,” “not comprise,” or “exclude” the particular components. Similarly, throughout the description, where processes and methods are described as not including particular steps or features, it should be understood that the processes and methods may also “not comprise,” “exclude,” or “not have” the particular steps or features.

[0270] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0271] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0272] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or”in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0273] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the method remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0274] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.EXAMPLES

[0275] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1

[0276] This example describes the persistence and efficacy of MuSK-CAR T cells in the treatment of myasthenia gravis in subjects that did not receive any lymphodepleting preconditioning regimen.

[0277] Rationale

[0278] Muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG) is a rare but potentially severe disease, in which patients develop pathogenic autoantibodies that specifically target the MuSK protein in the neuromuscular junction. Compared with the more common type of MG, in which the acetylcholine receptor (AChR) is targeted, MuSK MG more commonly involves the bulbar muscles that affect speaking, chewing, and swallowing and respiratory muscles. The disease can be severe and rapidly progressive, sometimes leading to respiratory crisis, and may also lead to permanent muscle atrophy over time. In addition, there are fewer treatment options for MuSK MG, as patients respond poorly to acetylcholinesterase inhibitors and are not candidates for treatment with complement inhibitors such as eculizumab. Therefore, the standard treatment approach is to use therapeutics that generally suppress the immune system.

[0279] Study Design

[0280] Human MG patients who were seropositive for anti-MuSK antibodies were selected for treatment with an autologous CAR T cell therapy. The adult subjects had a confirmed diagnosis of MG with Myasthenia Gravis Foundation of America (MGFA) Clinical Classification I-IVa, were positive for anti-MuSK antibodies and negative for anti-AChR antibodies. In a pretreatment period, PMBCs were obtained from each patient via leukapheresis, and T-cells were activated and transduced with a lentiviral MuSK-CAR vector using standard methods. The lentiviral vector encoded a MuSK-CAR comprising an extracellular binding domain comprising a MuSK autoantigen, a CD 8 a transmembrane domain, a 4- IBB costimulatory domain, and a CD3^ signaling domain (FIGURE 1C). The MuSK autoantigen, which enables binding to MuSK-autoantibody-producing B cells, corresponded to the full-length extracellular domain of MuSK and comprised the amino acid sequence of SEQ ID NO: 54. (CARs comprising extracellular autoantigens rather than scFvs can also be referred to herein as chimeric autoantigen receptors, i.e., CAARs.)

[0281] The transduced T cells were expanded ex vivo, and the percentage of MuSK-CAR T cells in the manufactured product (“MP”) was quantified via flow cytometry (FIGURE 3, left column, top two graphs). A total dose of 5x108CAR T cells (“the Al cohort”) or 2.5 x 109CAR T cells (“the A2 cohort”) was administered to each subject via intravenous (IV) infusion. Each subject was monitored post- infusion for CAR T cell persistence, myasthenia gravis clinical scores, autoantibody levels, and cytokine levels.

[0282] In contrast to methods of treating autoimmune diseases with CAR T cells previously disclosed in the art, the MG patients were not administered a lymphodepleting preconditioning regimen prior to, or concurrently with, the CAR T cell infusion. Lymphodepletion is conventionally understood to be important for enabling CAR T cell persistence and survival in the infused subject because, e.g., it increases levels of homeostatic cytokines such as IL-7 and IL- 15 in the subject, and eliminates endogenous immune cells that would compete for said cytokines. Additionally, no cytokines were administered to the patients during or following infusion of the CAR T cells, and the cells were not engineered to ectopically express any cytokines or cytokine-signaling proteins (or any proteins other than the CAR construct).

[0283] Persistence and Expansion

[0284] The CAR T cell population successfully expanded in the patients post-infusion, even though the patients had not been administered a lymphodepleting preconditioning regimen. Briefly, PBMC and serum samples were obtained from the patients at timepoints up to 29 days post- infusion. The persistence of engineered T-cells was assessed using qPCR for the vector, and using flow cytometry to quantify CAR-expressing T cells. IFNy serum levels were determined using an IFNy multiplex immunoassay (Meso Scale Discovery). Results were quantified as vector copies per pg of DNA (FIGURE 2A) and as the number of MuSK-CAR T cells per pl of blood (FIGURE 2C). FIGURES 2B and 2D are bar graphs depicting the area under the curve (AUC) for the data shown in FIGURES 2A and 2C, respectively, through Day 29. For the Al cohort (5 x 108MuSK-CAR T cells), results were also quantified as the percentage of T cells expressing MuSK-CAR over time (FIGURE 3).

[0285] As shown in FIGURES 2A-2D, the MuSK-CAR T cells persisted at high levels in vivo for an extended duration, and up to 29 days post-infusion. The observed kinetics were similar to kinetics observed in aCD19-CAR T cell therapies for the treatment of hematologic malignancies. As shown in FIGURE 3, at peak expansion, 4.9% of subject Al-l’s T cells and 18% of subject Al-2’s T cells expressed the MuSK-CAR construct. Elevated levels of IFNy were also detected in several subjects (e.g., Al -2, A2-2, and A2-3) (FIGURE 4).

[0286] Autoantibody Levels and Clinical Scores

[0287] Autoantibody levels decreased in several MG patients following administration of the MuSK-CAR T cell therapy, even though patients were not administered lymphodepleting preconditioning. The subjects’ anti-MuSK autoantibody levels were monitored for about four months post-infusion with MuSK-CAR T cells. Autoantibody levels in serum samples were determined via ELISA, and results were quantified as nmol / L (FIGURE 5). As shown in FIGURE 5, MuSK autoantibody levels in several of the patients (patients Al-1, A2-2, and A2- 3) decreased post-infusion.

[0288] Certain myasthenia gravis clinical scores improved in several subjects following administration of the MuSK CAR T cells without lymphodepleting preconditioning. The subjects’ MG clinical scores were monitored for up to six months following infusion with the MuSK-CAR T cells. Clinical scores were measured using the Myasthenia Gravis Compositescale (MG Composite, FIGURE 6A, see Burns etal. (2010) NEUROLOGY 74(18): 1434-40), the Myasthenia Gravis activities of daily living profile (MG-ADL, FIGURE 6B, see Wolfe et al. (1999) NEUROLOGY 52 (7): 1487-89), the improved 15-item myasthenia gravis quality of life scale (MG-QOL 15r, FIGURE 6C, see Burns et al.) , and the quantitative Myasthenia Gravis scoring system (QMG, FIGURE 6D, see Jaretzki et al. (2000) NEUROLOGY 55(1): 16-23). As summarized in FIGURES 6A-6D, several subjects (e.g., Al-1) experienced improvements in each of the MG Composite, MG-ADL, MG-QOL 15r, and QMG clinical scores.

[0289] Comparison with Dsg3-CAR T Treatments

[0290] The persistence of MuSK-CAR T cells from the subjects that were administered MuSK-CAR T cells without lymphodepleting preconditioning were compared with historical data from subjects who were administered a different CAR construct: a CAR comprising a Dsg3 autoantigen (comprising the amino acid sequence of SEQ ID NO: 100), a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3(j signaling domain (also referred herein as CAB-101). Subjects with active, anti-DSG3, mucosal-dominant PV were intravenously administered a total dose of 5 x 108(Cohort “A3”) or 2.5 x 109(Cohort “A4”) Dsg3-CAR T cells without any lymphodepleting preconditioning regimen or cytokines (see the clinical trial plan as set forth in ClinicalTrials.gov ID No. NCT04422912). Persistence of engineered Dsg3-CAR T cells was assessed using qPCR for the vector, and using flow cytometry to quantify CAR- expressing T cells. As shown in FIGURES 2A-2D, Dsg3-CAR T cells did not effectively persist in vivo in the absence of preconditioning. MuSK-CAR T cells administered without any lymphodepleting preconditioning regimen exhibited superior in vivo persistence than comparable treatments with Dsg3-CAR T cells.

[0291] An additional cohort of PV patients was subsequently administered a total dose of 2.5 x 109Dsg3-CAR T cells using the same protocol, except the patients were also administered cyclophosphamide, fludarabine, and IVIg prior to administration of the Dsg3-CAR-T cells. The addition of a lymphodepleting pretreatment regimen did not provide serologic or clinical improvement, and did not deeply deplete B-cell levels in patients. These results demonstrate that, although the Dsg3-CAR construct itself is an ineffective CAR construct (irrespective of whether the Dsg3-CAR T cells are administered with preconditioning), lymphodepletingpreconditioning is unnecessary when autoimmune patients are treated with a functional and effective B-cell-targeted CAR-T therapeutic (e.g., MuSK-CAR T cells).Example 2

[0292] This example describes a trial to test the persistence and efficacy of MuSK-CAR T cells in the treatment of myasthenia gravis in additional subjects that did not receive a lymphodepleting preconditioning regimen.

[0293] Adult subjects who have a confirmed diagnosis of MG with MGFA Clinical Classification I-Iva, who are positive for anti-MuSK antibodies, and who are negative for anti- AChR antibodies are selected for treatment with the MuSK-CAR T cell therapy as in Example 1. PMBCs are obtained from each subject via leukapheresis, and MuSK-CAR T cells (engineered to express the same MuSK-CAR T construct described in Example 1) are manufactured as described in the same manner as described in Example 1. The MG patients are not administered a lymphodepleting preconditioning regimen prior to, or concurrently with, the CAR T cell infusion. Additionally, no cytokines are administered to the patients during or following infusion of the CAR T cells, and the cells are not engineered to ectopically express any cytokines or cytokine-signaling proteins (or any proteins other than the CAR construct).

[0294] The subjects are administered a dose of 5xl08, 2.5xl09, or from 5xl09to 7.5xl09CAR T cells. The subjects are monitored for about 36 months post-treatment. CAR T cell persistence in the subjects is monitored by measuring MuSK-CAR markings per cell number in subjects via qPCR, as described in Example 1. Myasthenia gravis clinical scores are monitored for each subject post-treatment, including MG Composite, MG-ADL, MG-QOL 15r, and QMG, as described in Example 1. MuSK-autoantibody levels and cytokine levels (e.g., serum IFNy levels) are also monitored in each subject post- infusion, as described in Example 1.

[0295] It is contemplated that the administered MuSK-CAR T cells can effectively expand and persist in the subjects, even though the subjects are not administered lymphodepleting preconditioning.Example 3

[0296] This example describes a phase 1 / 2 study of autologous CD19-specific CAR T cells in subjects with pemphigus vulgaris (PV), wherein the method optionally does not includeI l ladministration of a lymphodepleting preconditioning regimen. The study includes both mucosal- dominant PV (mPV; anti-DSG3 antibody positive) and mucocutaneous PV patients (mcPV; anti- DSG3 and anti-DSGl antibody positive).Background

[0297] Pemphigus vulgaris (PV) is a rare, life-threatening autoimmune blistering disease caused by autoantibodies to skin cell adhesion proteins known as desmogleins (DSGs) (Kasperkiewicz etal. (2017) NAT. REV. Dis. PRIMERS 3: 17026). There are two major clinical subtypes of PV, each with a characteristic autoantibody profile: mucosal-dominant PV, which results in painful blistering of the oropharyngeal, esophageal, nasal, genital, rectal, and conjunctival mucosae and is caused by autoantibodies to DSG3; and mucocutaneous PV, the more common subtype, associated with painful blistering of mucosae and skin, and caused by autoantibodies to both DSG3 and DSG1. The clinical and histologic site of blister formation in PV can be explained by the expression pattern of DSGs within epithelial tissues (Mahoney et al. (1999) J. CLIN. INVEST. 103(4): 461-468). Anti-DSG antibodies in PV have been shown to be both necessary and sufficient for blister formation. PV is associated with considerable morbidity and mortality and, left untreated, PV is frequently fatal.Investigation Product

[0298] The investigational product, CAB-001, is an autologous CD 19-targ eting chimeric antigen receptor (CAR) T cell. The CAR comprises a fully human scFv which specifically binds CD 19, a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3(j signaling domain. The anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 9, and the CAR construct comprises the amino acid sequence of SEQ ID NO: 23.Objectives and Endpoints

[0299] An objective of this study is to evaluate the safety and tolerability of the CAB-001 regimen in subjects with PV over the course of 28 days, measured by incidence of adverse events (AEs) occurring within 28 days after CAB-001 infusion.

[0300] An additional objective of this study is to evaluate the safety and tolerability of the CAB-001 regimen in subjects with PV over 156 weeks, measured by AEs, vital signs, physical examination, and clinical laboratory tests occurring within 156 weeks after CAB-001 infusion.

[0301] An additional objective of this study is to evaluate the effect of CAB-001 regimen on white blood cell counts, T cell counts, B cell counts, NK cell counts, and myeloid populations, measured from baseline in white blood cell counts with differential, as well as T, B, and NK cell counts and immunophenotypic sub-populations following CAB-001 infusion.

[0302] An additional objective of this study is to evaluate CAB-001 persistence and kinetics in vivo following infusion, measured by the number and percentage of CAB-001 -positive cells in peripheral blood of subjects over time.

[0303] An additional objective of this study is to evaluate the effect of the CAB-001 regimen on PV serology, measured as the change in serum anti-DSG3 and anti-DSGl antibody titer by ELISA (and anti-keratinocyte antibodies by indirect immunofluorescence (IIF)) over 156 weeks.

[0304] An additional objective of this study is to evaluate the effect of the CAB-001 regimen on PV disease activity, measured as the absolute and percent change in disease activity by Pemphigus Disease Area Index (PDAI) over 156 weeks.

[0305] An additional objective of this study is to evaluate the time required to achieve disease response following CAB-001 infusion, measured as (1) the time to complete remission off therapy from the last infusion, determined by a PDAI activity score of 0 for at least 2 months; (2) the time to complete remission on minimal therapy (Murrell et al. (2008) J. AM. ACAD. DERMATOL. 58(6): 1043-1046) from the last infusion, determined by a PDAI activity score of 0 for at least 2 months; and / or (3) the time to partial remission off therapy from the last infusion, defined as transient lesions that heal within 1 week while the patient is off therapy for at least 2 months.

[0306] An additional objective of this study is evaluate the effect of the CAB-001 regimen on concomitant steroid use and other PV-related therapy, measured as the change in dose of concomitant corticosteroids and oral adjuvant immunosuppressive therapies (e.g., mycophenolate mofetil, azathioprine, methotrexate, cyclophosphamide, and cyclosporine) over 156 weeks, as applicable.

[0307] An additional objective of this study is to evaluate the effect of the CAB-001 regimen on patient-reported outcomes, measured as the change in patient-reported outcomes ABQOL and ABSIS over 156 weeks.

[0308] An additional objective of this study is to evaluate the effect of the CAB-001 regimen on disease remission, measured as (1) the proportion of subjects achieving complete remission off therapy, determined by a PDAI activity score of 0 for at least 2 months; (2) the proportion of subjects achieving complete remission on minimal therapy (Murrell et al., 2008) determined by a PDAI activity score of 0 for at least 2 months; and / or (3) the proportion of subjects achieving partial remission off therapy, defined as transient lesions that heal within 1 week for at least 2 months.Study Design

[0309] This study is designed to evaluate the safety, tolerability, and efficacy of an anti-CD19 CAR T therapeutic regimen in PV subjects. Any subject who receives CAB-001 is followed after infusion for 156 weeks for safety and efficacy. The study will include subjects who are diagnosed with PV and are anti-DSG3 antibody positive with or without anti-DSGl antibody (mPV and mcPV).

[0310] At least two subjects are enrolled into each dose / conditioning cohort. Each subject undergoes leukapheresis, and the CAB-001 CD 19 CAR T cell product for the subject is manufactured by transducing the subject’s T cells with a vector encoding the anti-CD19 CAR. The manufactured product is formulated and cryopreserved prior to use. Subjects receive a single dose of CAB-001 and are evaluated during an initial 28-day post-treatment assessment period.

[0311] Patients are administered the genetically modified CAR T cells according to the dosing scheme summarized in TABLE 4. Briefly, in contrast to methods of treating autoimmune diseases with anti-CD19 CAR T cells previously disclosed in the art, certain cohorts of patients (Cl, C2, C3 in TABLE 4) are not administered a lymphodepleting preconditioning regimen prior to, or concurrently with, the CAR T cell infusion. Additionally, no cytokines are administered to the patients in any cohort during or following infusion of the CAR T cells, and the cells are not engineered to ectopically express any cytokines or cytokine-signaling proteins in any cohort.

[0312] Patients in an optional fourth cohort (C4 in TABLE 4) are administered a lymphodepleting preconditioning regimen. Specifically, patients in Cohort C4 are administered one dose of 1,000 mg / m2cyclophosphamide and three doses of 25 mg / m2fludarabine in the dayspreceding administration of the CAR T cells. Fludarabine is administered at a dose of 25 mg / m2intravenously on 3 consecutive days, Day -5, Day -4, and Day -3, before the initiation of CAB- 001 on Day 1. Cyclophosphamide is administered at a dose of 1,000 mg / m2intravenously on Day -3 before the initiation of CAB-001.

[0313] A dose of CAR T cells is intravenously administered to each patient as summarized in TABLE 4. Briefly, patients in Cohorts Cl, C2, and C3 are administered the genetically modified autologous anti-CD19 CAR T cells at a dose of 1 x 106cells / kg, 3 x 106cells / kg, and 1 x 107cells / kg, respectively. Patients in Cohort C4 are administered a dose of 1 x 106cells / kg.TABLE 4. Summary of Dosing Plan

[0314] The first two subjects in each cohort will be monitored as an inpatient for a minimum of 4 days after CAB-001 infusion. Subsequent visits may occur on Days 5, 8, 15, 22, and 29 after infusion. In addition to routine chemistry and hematology tests, lab tests for PK sampling, coagulation panel, CRS labs, serum cytokine assessment, and CAR T assessment are performed through Day 29. After the Day 29 post-infusion evaluation, subjects undergo evaluations approximately every 4 weeks in the first year, approximately every 12 weeks in the second year, and approximately once every 26 weeks in the third year. Patients may be monitored for up to 15 years post-infusion, as appropriate.

[0315] Whole blood samples are collected for measurement of CAR T cell persistence. In addition, research- based assessments are conducted on the CAR T cells (e.g., flow cytometry assessment and single cell RNA sequencing), B cells (e.g., flow cytometric analysis and B-cell receptor sequencing to define B cell repertoire), and soluble biomarker measurement (e.g., autoantibody and cytokine profiling).

[0316] Peripheral blood is collected to evaluate the expansion and persistence of CAB-001 cells after infusion through 156 weeks and can continue, as appropriate, until 2 consecutive samples are negative for CAB-001 cells.Inclusion / Exclusion Criteria

[0317] Inclusion criteria for the study are: (1) able to provide informed consent; (2) at least 18 years of age; have an Eastern Cooperative Oncology Group (ECOG) performance status 0-1; (4) have a diagnosis of PV based on prior or screening biopsy for histology AND prior positive DSG3 ELISA, IIF, and / or DIF; (5) have PV that has previously been inadequately managed by, or refractory to, or relapsed after, or with contraindications to or intolerance of at least one prior standard therapy (corticosteroids, mycophenolate mofetil, azathioprine, rituximab, plasmapheresis, intravenous immunoglobulin, methotrexate, cyclophosphamide, and cyclosporine); if on mycophenolate mofetil, azathioprine, methotrexate, cyclophosphamide, or cyclosporine, the dose needs to be stable for at least 12 weeks and if no longer on these medications, they need to have been discontinued for at least 12 weeks prior to screening; (6) have active disease at screening: PV PDAI score > 9 or PV PDAI Mucous Membrane score > 4; (7) have DSG3 ELISA >125% of the negative cutoff value of the assay at Screening; (8) in the opinion of the investigator, additional systemic therapy is warranted, and the subject is a reasonable candidate for CAR T therapy; (9) have received all currently recommended vaccinations per Centers for Disease Control and Prevention (CDC) or institutional guidelines for immunocompromised individuals before or during Screening, including COVID-19 / severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if applicable; (9a) live vaccines must be administered at least 30 days prior to the Pre-Infusion Visit, and (9b) non-live vaccines should be administered to subjects at least 2 weeks prior to the start of study drug infusion and, if possible, at least 2 weeks prior to leukapheresis; (10) have clinical stability by vital signs assessment at the time of screening, including: a. Systolic blood pressure >90 mmHg and <170 mmHg, b. Diastolic blood pressure >55 mmHg and <105 mmHg, c. Pulse >50 and <110 beats per minute, d. Respiratory rate >12 and <20 breaths per minute, e. Afebrile; (11) women of reproductive potential must agree to use 2 acceptable methods of birth control from Screening to a minimum of 52 weeks after the CAB-001 infusion, where acceptable contraception methods include intrauterine device, hormone-based contraception, and barrier method (e.g., condom, diaphragm, or cervical cap) with spermicide; (12) sexually active men who are reproductivelycompetent, including vasectomized men (who do not have confirmation of infertility by laboratory testing), who are to receive CAB-001 are required to use condoms with spermicide from Screening until at least 52 weeks after CAB-001 infusion, and males of reproductive potential must also agree not to donate sperm from Screening to at least 52 weeks after the CAB- 001 infusion.

[0318] Exclusion criteria for the study are: (1) have paraneoplastic pemphigus or active malignancy (not including non-melanoma skin cancer); (2) have oral prednisone (or corticosteroid equivalent) dose >0.25 mg / kg / d; if on prednisone (or corticosteroid equivalent), the dose must be stable for at least 28 days prior to Screening; (3) have received rituximab or other anti-CD20 or anti-CD 19 therapies in last 12 months unless anti-DSG3 antibody titers have recently increased or PV symptoms have recently worsened objectively during a period after rituximab treatment and prior to Screening; (4) have had plasmapheresis or intravenous immunoglobulin (IVIg) infusion within 2 weeks of Screening; (5) have had investigational therapy in the last 3 months; (6) have contraindications to leukapheresis; (7) have Absolute lymphocyte count <500 / pl at Screening; (8) have positive human immunodeficiency virus (HIV), hepatitis C virus (HCV) antibody, or hepatitis B surface antigen test, or evidence of active or chronic tuberculosis at Screening; (9) have active infection requiring medical intervention; (10) have autoimmune disorder other than PV requiring immunosuppressants; (11) have New York Heart Association Class III or IV heart failure (see Appendix 2), unstable angina, or a history of recent (within 6 months) myocardial infarction or sustained (>30 seconds) ventricular tachyarrhythmias; (12) have organ failure or multi-organ dysfunction; (13) are pregnant or lactating; (14) have any medical / psychiatric condition that, in the opinion of the investigator, may interfere with interpretation of subject safety, evaluation of the investigational product, or protocol compliance; (15) are unable or unwilling to comply with protocol.

[0319] It is contemplated that the autologous CD19-CAR T cell therapy described in this example can be effective to treat PV in the absence of lymphodepleting preconditioning.Example 4

[0320] This example describes treating an autoimmune disease in a subject in need thereof by administering T cells engineered to express a CAR, wherein the method does not include administration of a lymphodepleting preconditioning regimen.

[0321] T cells are obtained from a subject having an autoimmune disease (for example, an autoimmune disease selected from (SLE), lupus nephritis, SLE with anti-dsDNA antibodies, pemphigus vulgaris (PV), mucosal PV, mucocutaneous PV, myasthenia gravis (MG), MuSK- associated MG, AChRMG, myositis juvenile myositis, membranous nephropathy, antisynthetase syndrome, dermatomyositis, immune mediated necrotizing myopathy, multiple sclerosis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, anti- NMDA Receptor encephalitis, Lambert-Eaton syndrome, pemphigus foliaceus, epidermolysis bullosa acquisita, bullous pemphigoid, Goodpasture’s syndrome, rheumatoid arthritis, systemic sclerosis, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, immune thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, type 1 diabetes, Grave’s disease, and Hashimoto’s disease). The T cells are transduced with a vector encoding a CAR that specifically binds a B-cell surface protein, e.g., an anti-CD19 CAR comprising a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3(j intracellular domain, e.g., a CAR comprising the amino acid sequence of SEQ ID NO: 23. The transduced T cells are expanded and, optionally, cryopreserved.

[0322] In contrast to methods of treating autoimmune diseases with CAR T cells previously disclosed in the art, the patients are not administered a lymphodepleting preconditioning regimen prior to, or concurrently with, the CAR T cell infusion. Additionally, no cytokines are administered to the patients during or following infusion of the CAR T cells, and the cells are not engineered to ectopically express any cytokines or cytokine-signaling proteins.

[0323] A control subject or control group may instead be administered a standard or reduced lymphodepleting preconditioning dose prior to and / or concurrently with infusion, e.g., a total dose of 1000 mg / m2cyclophosphamide and a total dose of 75-90 mg / m2fludarabine.

[0324] A dose of CAR T cells (e.g., from IxlO6to IxlO7cells / kg bodyweight) is intravenously administered to the subject. The subject is monitored post-infusion for CAR T cell levels and persistence post-infusion, B-cell levels, white blood cell count, and for autoimmune disease markers (e.g., autoantibody levels).

[0325] It is contemplated that the autologous CD19-CAR T cell therapy described in this example can be effective to treat the autoimmune disease in the absence of lymphodepleting preconditioning.INCORPORATION BY REFERENCE

[0326] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0327] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning ...

Claims

WHAT IS CLAIMED IS:

1. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain comprising a binding domain that binds a B-cell surface protein, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine, wherein the treatment establishes a population of genetically modified immune cells in the subject, and(a) the abundance of the DNA encoding the CAR reaches at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 1,000 copies per microgram of PBMC genomic DNA for at least 5 days; and / or(b) the population of genetically modified immune cells is established in the subject at a concentration of at least 100 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 5 days.

2. The method of claim 1, wherein the DNA encoding the CAR remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

3. The method of claim 1 or 2, wherein the abundance of the DNA encoding the CAR reaches at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 20,000, or at least 50,000 copies per microgram of genomic DNA in PBMCs harvested from the subject.

4. The method of any one of claims 1-3, wherein the abundance of the DNA encoding the CAR reaches at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 3,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

5. The method of any one of claims 1-4, wherein the abundance of the DNA encoding the CAR reaches at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 5,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

6. The method of any one of claims 1-5, wherein the abundance of the DNA encoding the CAR reaches at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 10,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

7. The method of any one of claims 1-6, wherein the genetically modified immune cell is administered to the subject on Day 0, and the DNA encoding the CAR remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, from Day 5 to Day 21, or from Day 5 to Day 29.

8. The method of claim 7, wherein the DNA encoding the CAR remains at an abundance of at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, from Day 5 to Day 21, or from Day 5 to Day 29.

9. The method of claim 7 or 8, wherein the DNA encoding the CAR remains at an abundance of at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, from Day 5 to Day 21, or from Day 5 to Day 29.

10. The method of any one of claims 7-9, wherein the DNA encoding the CAR remains at an abundance of at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, from Day 5 to Day 21, or from Day 5 to Day 29.

11. The method of any one of claims 1-10, wherein the abundance of the DNA encoding the CAR in PBMCs harvested from the subject is measured by quantitative PCR (qPCR).

12. The method of any one of claims 1-11, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

13. The method of any one of claims 1-12, wherein the population of genetically modified immune cells in the subject is established at a concentration of at least 200, at least 300, at least 400, at least 500 cells per microliter of blood.

14. The method of any one of claims 1-13, wherein the population of genetically modified immune cells in the subject is established at a concentration of at least 200 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 200 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

15. The method of any one of claims 1-14, wherein the population of genetically modified immune cells is established in the subject at a concentration of at least 300 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

16. The method of any one of claims 1-15, wherein the genetically modified immune cell is administered to the subject on Day 0, and the population of genetically modified immune cellspersists in the subject at a concentration of at least 100 cells per microliter of blood from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

17. The method of claim 16, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 200 cells per microliter of blood from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

18. The method of claim 16 or 17, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

19. The method of any one of claims 1-18, wherein the concentration of genetically modified immune cells in the blood of the subject is measured by flow cytometry.

20. The method of any one of claims 1-19, wherein the subject has not received a dose of cyclophosphamide and / or fludarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months prior to administration of the genetically modified immune cell.

21. The method of any one of claims 1-20, wherein the method does not include administering a chemotherapeutic agent to the subject before or during administration of the genetically modified immune cell.

22. The method of any one of claims 1-21, wherein the method does not include treating the subject with radiation therapy before or during administration of the genetically modified immune cell.

23. The method of any one of claims 1-22, wherein the method does not include exposing the subject to total lymphatic irradiation before or during administration of the genetically modified immune cell.

24. The method of any one of claims 1-23, wherein the genetically modified immune cell is not engineered to ectopically express a cytokine-pathway signaling protein.

25. The method of any one of claims 1-24, wherein the genetically modified immune cell is not engineered to ectopically express a cytokine receptor.

26. The method of any one of claims 1-25, wherein the genetically modified immune cell is not engineered to ectopically express a protein (e.g., a recombinant protein) other than the CAR.

27. The method of any one of claims 1 -26, wherein the genetically modified immune cell is not transfected with an mRNA prior to being administered to the subject.

28. The method of any one of claims 1-27, wherein the method does not include administering a recombinant cytokine to the subject before or during administration of the genetically modified immune cell.

29. The method of any one of claims 1-28, wherein the B-cell surface protein is CD 19.

30. The method of any one of claims 1-29, wherein the extracellular binding domain comprises an antigen-binding site that specifically binds the B-cell surface protein, the antigenbinding site comprising a heavy chain variable domain (VH) comprising complementarity determining regions CDRHI, CDRH2, and CDRJB and a light chain variable domain (VL) comprising complementarity determining regions CDRLI, CDRL2, and CDRLS.

31. The method of claim 30, wherein the antigen-binding site is humanized or fully human.

32. The method of claim 30 or 31, wherein:(i) the CDRHI, CDRH2, and CDRHS comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the CDRLI, CDRL2, and CDRLS comprise the amino acid sequences of SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively;(ii) the CDRHI, CDRH2, and CDRH3 comprise the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, and the CDRLI, CDRL2, and CDRLS comprise the amino acid sequences of SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively; or(iii) the CDRHI, CDRH2, and CDRH3 comprise the amino acid sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively, and the CDRLI, CDRL2,and CDRLS comprise the amino acid sequences of SEQ ID NO: 106, GAS, and SEQ ID NO: 47, respectively.

33. The method of any one of claims 30-32, wherein:(i) the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 4 and 8, respectively;(ii) the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 13 and 17, respectively; or(iii) the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 44 and 48, respectively.

34. The method of any one of claims 30-33, wherein:(i) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 4 and 8, respectively;(ii) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 13 and 17, respectively; or(iii) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 44 and 48, respectively.

35. The method of any one of claims 30-34, wherein the antigen-binding site is present in an scFv.

36. The method of claim 35, wherein the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51.

37. The method of claim 35 or 36, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51.

38. The method of any one of claims 1-28, wherein the B-cell surface protein comprises a MuSK autoantibody.

39. The method of claim 38, wherein the extracellular binding domain comprises a MuSK autoantigen.

40. The method of claim 39, wherein the MuSK autoantigen is a full-length MuSK autoantigen.

41. The method of claim 39 or 40, wherein the MuSK autoantigen comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

42. The method of any one of claims 39-41, wherein the MuSK autoantigen comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

43. The method of any one of claims 1-42, wherein the extracellular binding domain does not comprise a Dsg3 autoantigen.

44. The method of any one of claims 1-43, wherein the extracellular binding domain does not comprise a Dsg autoantigen.

45. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a genetically modified immune cell comprising a DNA encoding a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain comprising an antigen-binding site that specifically binds a B-cell surface protein, wherein the method does not include administering to the subject either (i) a lymphodepleting preconditioning regimen comprising administering either cyclophosphamide or fludarabine, or (ii) a purified cytokine or a cell engineered to ectopically express a cytokine.

46. The method of claim 45, wherein the treatment establishes a population of genetically modified immune cells in the subject.

47. The method of claim 46, wherein the abundance of the DNA encoding the CAR reaches at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 1,000 copies per microgram of PBMC genomic DNA for at least 5 days.

48. The method of claim 47, wherein the DNA encoding the CAR remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

49. The method of any one of claim 46-48, wherein the abundance of the DNA encoding the CAR reaches at least 2,000, at least 3,000, at least 4,000, at least 5,000, or at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject.

50. The method of any one of claims 46-49, wherein the abundance of the DNA encoding the CAR reaches at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 3,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

51. The method of any one of claims 46-50, wherein the abundance of the DNA encoding the CAR reaches at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 5,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

52. The method of any one of claims 46-51, wherein the abundance of the DNA encoding the CAR reaches at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject, and remains at an abundance of at least 10,000 copies per microgram of PBMC genomic DNA for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 28, or at least 29 days.

53. The method of any one of claims 46-52, wherein the genetically modified immune cell is administered to the subject on Day 0, and the DNA encoding the CAR remains at an abundance of at least 1,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21 .

54. The method of claim 53, wherein the DNA encoding the CAR remains at an abundance of at least 3,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

55. The method of claim 53 or 54, wherein the DNA encoding the CAR remains at an abundance of at least 5,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

56. The method of any one of claims 53-55, wherein the DNA encoding the CAR remains at an abundance of at least 10,000 copies per microgram of genomic DNA in PBMCs harvested from the subject from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

57. The method of any one of claims 46-56, wherein the abundance of the DNA encoding the CAR in PBMCs harvested from the subject is measured by quantitative PCR (qPCR).

58. The method of claim 46, wherein the population of genetically modified immune cells is established in the subject at a concentration of at least 100, at least 200, at least 300, at least 400, or at least 500 cells per microliter of blood.

59. The method of claim 46 or 58, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 5 days.

60. The method of any one of claims 46 or 58-59, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

61. The method of any one of claims 46 or 58-60, wherein the population of genetically modified immune cells in the subject is established at a concentration of at least 200 cells per microliter of blood, and the population of genetically modified immune cells persists in thesubject at a concentration of at least 200 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

62. The method of any one of claims 46 or 58-61, wherein the population of genetically modified immune cells in the subject is established at a concentration of at least 300 cells per microliter of blood, and the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood for at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 days.

63. The method of any one of claims 46 or 58-62, wherein the genetically modified immune cell is administered to the subject on Day 0, and the population of genetically modified immune cells persists in the subject at a concentration of at least 100 cells per microliter of blood from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

64. The method of claim 63, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 200 cells per microliter of blood from Day 5 to Day 10, from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

65. The method of claim 63 or 64, wherein the population of genetically modified immune cells persists in the subject at a concentration of at least 300 cells per microliter of blood from Day 5 to Day 12, from Day 5 to Day 14, or from Day 5 to Day 21.

66. The method of any one of claims 46-65, wherein the concentration of genetically modified immune cells in the blood of the subject is measured by flow cytometry.

67. The method of any one of claims 45-66, wherein the subject has not received a preconditioning regimen that includes cyclophosphamide and / or fludarabine for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months prior to administration of the genetically modified immune cell.

68. The method of any one of claims 45-67, wherein the method does not include administering a chemotherapeutic agent to the subject before or during administration of the genetically modified immune cell.

69. The method of any one of claims 45-68, wherein the method does not include treating the subject with radiation therapy before or during administration of the genetically modified immune cell.

70. The method of any one of claims 45-69, wherein the method does not include exposing the subject to total lymphatic irradiation before or during administration of the genetically modified immune cell.

71. The method of any one of claims 45-70, wherein the genetically modified immune cell is not engineered to ectopically express a cytokine-pathway signaling protein.

72. The method of any one of claims 45-71, wherein the genetically modified immune cell is not engineered to ectopically express a cytokine receptor.

73. The method of any one of claims 45-72, wherein the genetically modified immune cell is not engineered to ectopically express a protein (e.g., a recombinant protein) other than the CAR.

74. The method of any one of claims 45-73, wherein the genetically modified immune cell is not transfected with an mRNA prior to being administered to the subject.

75. The method of any one of claims 45-74, wherein the method does not include administering a recombinant cytokine to the subject before or during administration of the genetically modified immune cell.

76. The method of any one of claims 45-75, wherein the B-cell surface protein is CD19.

77. The method of any one of claims 45-76, wherein the antigen-binding site comprises a heavy chain variable domain (VH) comprising complementarity determining regions CDRm, CDRH2, and CDRHS and a light chain variable domain (VL) comprising complementarity determining regions CDRLI, CDRL2, and CDRLS.

78. The method of claim 77, wherein the antigen-binding site is humanized or fully human.

79. The method of claim 77 or 78, wherein:(i) the CDRHI, CDRH2, and CDRJB comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the CDRLI, CDRL2, and CDRLS comprise the amino acid sequences of SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively;(ii) the CDRHI, CDRH2, and CDRHS comprise the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, and the CDRLI, CDRL2, and CDRLS comprise the amino acid sequences of SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively; or(iii) the CDRHI, CDRH2, and CDRH3 comprise the amino acid sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively, and the CDRLI, CDRL2, and CDRLS comprise the amino acid sequences of SEQ ID NO: 106, GAS, and SEQ ID NO: 47, respectively.

80. The method of any one of claims 77-79, wherein:(i) the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 4 and 8, respectively;(ii) the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 13 and 17, respectively; or(iii) the VH and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 44 and 48, respectively.

81. The method of any one of claims 77-80, wherein:(i) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 4 and 8, respectively;(ii) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 13 and 17, respectively; or(iii) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 44 and 48, respectively.

82. The method of any one of claims 77-81, wherein the antigen-binding site is present in an scFv.

83. The method of claim 82, wherein the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51.

84. The method of claim 82 or 83, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 9, 18, and 51.

85. The method of any one of claims 1-84, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain.

86. The method of claim 85, wherein the transmembrane domain comprises a CD8 alpha chain transmembrane domain.

87. The method of claim 86, wherein the CD8 alpha chain transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19.

88. The method of any one of claims 85-87, wherein the costimulatory domain comprises a 4- IBB intracellular domain.

89. The method of claim 88, wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 20.

90. The method of any one of claims 85-89, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

91. The method of claim 90, wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 21.

92. The method of any one of claims 85-91, wherein the CAR further comprises a hinge domain or linker interposed between the extracellular binding domain and the transmembrane domain.

93. The method of claim 92, wherein the hinge domain is a CD8 alpha chain hinge.

94. The method of claim 93, wherein the CD8 alpha chain hinge comprises the amino acid sequence of SEQ ID NO: 22.

95. The method of any one of claims 1-94, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 23 and 27.

96. The method of any one of claims 1-84, wherein the CAR further comprises a killer immunoglobulin-like receptor (KIR) transmembrane domain and a KIR cytoplasmic domain.

97. The method of any one of claims 1 -96, wherein the genetically modified immune cell is a T cell or an NK cell.

98. The method of claim 97, wherein the genetically modified immune cell is a T cell.

99. The method of claim 97 or 98, wherein the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a memory T cell, an alpha beta T cell, and a gamma delta T cell.

100. The method of any one of claims 97-99, wherein the T cell is a cytotoxic T cell.

101. The method of any one of claims 1-100, wherein the genetically modified immune cell is autologous to the subject.

102. The method of any one of claims 1-101, wherein the genetically modified immune cell is administered to the subject in a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier or excipient.

103. The method of any one of claims 1-102, wherein the method comprises administering the genetically modified immune cell to the subject at a dose from 1 x 105cells / kg to 1 x 108cells / kg.

104. The method of claim 103, wherein the dose is from 1 x 106cells / kg to 1 x 107cells / kg.

105. The method of any one of claims 1-104, wherein the DNA encoding the CAR is introduced into the genetically modified immune cell by a viral vector.

106. The method of claim 105, wherein the viral vector is a lentiviral vector or an adeno- associated viral vector.

107. The method of claim 106, wherein the viral vector is a lentiviral vector.

108. The method of any one claims 1-107, wherein the autoimmune disease is a B-cell- mediated autoimmune disease.

109. The method of any one of claims 1-108, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myasthenia gravis (MG), myositis, systemic sclerosis, multiple sclerosis, membranous nephropathy, and chronic immune demyelinating polyneuropathy.

110. The method of any one of claims 1-109, wherein the autoimmune disease is selected from the group consisting of lupus nephritis, SLE with anti-dsDNA antibodies, mucosal PV, mucocutaneous PV, MuSK-associated MG, AChR MG, anti-synthetase syndrome, dermatomyositis, and immune mediated necrotizing myopathy.

111. The method of any one of claims 1-110, wherein the autoimmune disease is MuSK- associated MG.

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