Methods and compositions for preparing genetically modified immune cells

Abstract

The invention relates generally to methods of preparing engineered T cells for adoptive cell therapy, including methods utilizing a whole blood sample isolated from a subject. The invention also relates generally to cell therapeutic compositions produced by such methods, as well as to therapeutic methods of using of such compositions.

Description

METHODS AND COMPOSITIONS FOR PREPARING GENETICALLY MODIFIED IMMUNE CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 644,757, filed May 9, 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 May 6, 2025, is named CBB-206WO_SL.xml and is 146,323 bytes in size.FIELD OF THE INVENTION

[0003] The invention relates generally to methods of preparing engineered T cells for use adoptive cell therapy, including methods utilizing primary T cells obtained from a whole blood sample isolated from a subject.BACKGROUND

[0004] 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, 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. 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 therapy induced clinical remission in 5 out of 5 patients with moderate to severe, refractory SLE (Mackensen et al. (2022) NAT. MED. 28: 2124-2132).

[0005] Despite the efficacy of CAR T cell therapy in treating autoimmune diseases and cancer, significant logistical barriers exist which limit patient access to cell therapy. Inparticular, the T cells used to manufacture CAR T cells are generally obtained from the patient by leukapheresis, which is typically carried out at an apheresis center by trained staff using specialized equipment. However, the distribution and capacity of apheresis centers, as well as the cost, duration, and invasiveness of the procedure, pose barriers to the continued expansion of CAR T therapies, including the use of CAR T therapy in the treatment of autoimmune diseases. Accordingly, in spite of the advancements made to date, there remains a need for new and useful methods of preparing CAR T cells.SUMMARY OF THE INVENTION

[0006] Leukapheresis is typically used to isolate immune cells from subjects for use in preparing genetically modified immune cells for adoptive cell therapy (ACT). However, access to apheresis centers presents a significant obstacle to the more widespread adoption of ACTs, such as CAR-T cell therapy. It has now been discovered that genetically modified T cells can be efficiently and effectively manufactured from primary T cells obtained from a whole blood sample isolated from a subject. Whole blood can be obtained from subjects through a simple blood draw, thereby eliminating the need for specialized apheresis centers and equipment.

[0007] Accordingly, in one aspect, the present disclosure provides a method of producing a composition of engineered T cells that express a chimeric receptor, the method comprising: (a) contacting a population of primary T cells with an activating agent, wherein the population of primary T cells has been obtained from a whole blood sample isolated from a subject, thereby generating a population of activated T cells; (b) introducing into the activated T cells a heterologous polynucleotide encoding the chimeric receptor, thereby generating a population of genetically modified T cells; (c) incubating the population of genetically modified T cells in a basal culture medium for at least two days, thereby generating a population of expanded T cells; and (d) harvesting the expanded T cells, thereby producing a composition of engineered T cells. In certain embodiments, the whole blood sample obtained from the subject comprises 50-300 mL of whole blood, for example, 50-250 mL of whole blood, e.g., 80-250 mL of whole blood.

[0008] PBMCs can be isolated from the whole blood sample using, for example, density gradient centrifugation or by adding a red-blood-cell lytic agent (e.g., water or ammonium- chloride-potassium (ACK) lysis buffer) to the whole blood sample.

[0009] In certain embodiments, the subject has an autoimmune disease. The autoimmune disease can be selected from, for example, the group consisting of systemic lupuserythematosus (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 SLE.

[0010] In certain embodiments of any of the foregoing methods, the population of primary T cells is contacted with the activating agent within 24, 48, or 72 hours of when the whole blood sample is isolated from the subject.

[0011] In certain embodiments of any of the foregoing methods, the method further comprises, prior to step (a): (I) obtaining a population of PBMCs isolated from the whole blood sample; and (II) enriching for CD4+ and CD8+ T cells in the population of PBMCs, thereby generating the population of primary T cells. Step (I) can comprise thawing a frozen sample comprising the population of PBMCs. Alternatively, the population of PBMCs is never frozen. For example, in certain embodiments, the population of PBMCs is stored at a temperature above 0 °C and below 22 °C. Step (II) can comprise positively selecting for CD4+ T cells and CD 8+ T cells. In certain embodiments, the population of primary T cells is frozen after being generated in step (II).

[0012] In certain embodiments of any of the foregoing methods, the method further comprises, prior to step (a), a step of (I) enriching for CD4+ and CD8+ T cells in the whole blood sample, thereby generating the population of primary T cells. The population of primary T cells can optionally be frozen after being generated in step (I).

[0013] In certain embodiments, the whole blood sample and / or the population of primary T cells is never frozen. For example, the whole blood sample and / or the population of primary T cells can be stored at a temperature above 0 °C and below 22 °C. In certain embodiments, the PBMCs are frozen after being isolated from the whole blood sample.

[0014] In certain embodiments of any of the foregoing methods, the activating agent comprises an antibody or an antigen-binding fragment thereof. The antibody or antigenbinding fragment thereof can be, e.g., humanized or fully human. In certain embodiments, the activating agent comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

[0015] In certain embodiments of any of the foregoing methods, the activating agent is bound to a bead, e.g., a magnetic bead. Alternatively, the activating agent can be bound to a polymeric nanomatrix.

[0016] In certain embodiments of any of the foregoing methods, the contacting in step (a) is carried out for at least 12 hours, e.g., for at least 24 hours. Step (a) and / or step (b) may optionally be carried out in a further basal culture medium. In certain embodiments, step (a) comprises seeding the population of primary T cells in the further basal culture medium at aconcentration of 5xl05to 5xl06cells per mL. The further basal culture medium may comprise, for example, IL-2, IL-7, and / or IL-15 (e.g., the combination of IL-7 and IL-15). In certain embodiments, step (b) comprises culturing the activated T cells in the further basal culture medium for at least 1 day or at least 2 days.

[0017] The heterologous polynucleotide can optionally be comprised in a viral vector, for example, a lentiviral vector or an adeno-associated viral vector, e.g., a lentiviral vector. In certain embodiments, the population of activated T cells is incubated with the viral vector at a multiplicity of infection (MOI) of 4 to 10, e.g., at an MOI of 7 to 9.

[0018] In certain embodiments of any of the foregoing methods, the basal culture medium comprises IL-2, IL-7, and / or IL- 15, for example, the combination of IL-7 and IL- 15. Additionally or alternatively, the basal culture medium may comprise, for example, human male AB serum (HABS).

[0019] In certain embodiments of any of the foregoing methods, step (c) is carried out in a bioreactor. Step (c) can comprise, for example, incubating the population of genetically modified T cells in a basal culture medium for at least three days, at least four days, at least five days, or at least six days.

[0020] In certain embodiments of any of the foregoing methods, the whole blood sample has not been subjected to leukapheresis.

[0021] In a related aspect, the present disclosure provides method of producing a composition of engineered T cells that express a chimeric receptor, the method comprising: (a) contacting a population of primary T cells with an activating agent, wherein the population of primary T cells has been obtained from a sample isolated from a subject, thereby generating a population of activated T cells; (b) introducing into the activated T cells a heterologous polynucleotide encoding the chimeric receptor, thereby generating a population of genetically modified T cells; (c) incubating the population of genetically modified T cells in a basal culture medium for at least two days, thereby generating a population of expanded T cells; and (d) harvesting the expanded T cells, thereby producing a composition of engineered T cells, wherein: (i) the subject has an autoimmune disease; and / or (ii) the population of primary T cells is contacted with the activating agent within 72 hours of when the sample was isolated from the subject. In certain embodiments, the sample is a whole blood sample. The whole blood sample obtained from the subject can comprise, for example, 50-300 mL of whole blood, e.g., 50-250 mL of whole blood or 80-250 mL of whole blood.

[0022] PBMCs can be isolated from the whole blood sample using, for example, density gradient centrifugation or by adding a red- blood-cell lytic agent (e.g., water or ammonium- chloride-potassium (ACK) lysis buffer) to the whole blood sample.

[0023] In certain embodiments, 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. For example, in certain embodiments, the autoimmune disease is SLE.

[0024] In certain embodiments, the population of primary T cells is contacted with the activating agent within 24, 48, or 72 hours of when the sample is isolated from the subject.

[0025] In certain embodiments, the method further comprises, prior to step (a): (I) obtaining a population of PBMCs isolated from the sample; and (II) enriching for CD4+ and CD8+ T cells in the population of PBMCs, thereby generating the population of primary T cells. Step (I) can comprise thawing a frozen sample comprising the population of PBMCs. Alternatively, the population of PBMCs is never frozen. For example, in certain embodiments, the population of PBMCs is stored at a temperature above 0 °C and below 22 °C. Step (II) can optionally comprise positively selecting for CD4+ T cells and CD8+ T cells. In certain embodiments, the population of primary T cells is frozen after being generated in step (II).

[0026] In certain embodiments, the method further comprises, prior to step (a), a step of (I) enriching for CD4+ and CD8+ T cells in the whole blood sample, thereby generating the population of primary T cells. The population of primary T cells can optionally be frozen after being generated in step (I). In certain embodiments, the whole blood sample and / or the population of primary T cells is never frozen. For example, the whole blood sample and / or the population of primary T cells can be stored at a temperature above 0 °C and below 22 °C. In certain embodiments, the PBMCs are frozen after being isolated from the whole blood sample.

[0027] In certain embodiments, the activating agent comprises an antibody or an antigenbinding fragment thereof. The antibody or antigen-binding fragment thereof can optionally be humanized or fully human. In certain embodiments, the activating agent comprises an anti-CD3 antibody and / or an anti-CD28 antibody. In certain embodiments, the activating agent is bound to a bead, e.g., a magnetic bead. Alternatively, the activating agent can be bound to a polymeric nanomatrix.

[0028] In certain embodiments, the contacting in step (a) is carried out for at least 12 hours, e.g., for at least 24 hours. Step (a) and / or step (b) may optionally be carried out in a further basal culture medium. In certain embodiments, step (a) comprises seeding the population of primary T cells in the further basal culture medium at a concentration of 5xl05to 5xl06cells per mL. The further basal culture medium may comprise, for example, IL-2, IL-7, and / or IL- 15 e.g., the combination of IL-7 and IL-15). In certain embodiments, step (b) comprises culturing the activated T cells in the further basal culture medium for at least 1 day or at least 2 days.

[0029] The heterologous polynucleotide can optionally be comprised in a viral vector, for example, a lentiviral vector or an adeno-associated viral vector, e.g., a lentiviral vector. In certain embodiments, the population of activated T cells is incubated with the viral vector at a multiplicity of infection (MOI) of 4 to 10, e.g., at an MOI of 7 to 9.

[0030] In certain embodiments, the basal culture medium comprises IL-2, IL-7, and / or IL-15, for example, the combination of IL-7 and IL-15. Additionally or alternatively, the basal culture medium may comprise, for example, human male AB serum (HABS).

[0031] In certain embodiments, step (c) is carried out in a bioreactor. Step (c) can comprise, for example, incubating the population of genetically modified T cells in the basal culture medium for at least three days, at least four days, at least five days, or at least six days.

[0032] In certain embodiments, the sample has not been subjected to leukapheresis.

[0033] In certain embodiments of any of the foregoing methods, at least 50%, at least 55%, at least 60%, or at least 65% of the population of expanded T cells expresses the chimeric receptor. Additionally or alternatively, the population of expanded T cells can comprise at least IxlO8, at least 2xl08, or at least 3xl08viable nucleated cells.

[0034] In certain embodiments of any of the foregoing methods, the chimeric receptor is a chimeric antigen receptor (CAR) comprising an extracellular binding domain comprising an antigen binding site that specifically binds a cell-surface protein, wherein the antigen-binding site comprises a heavy chain variable domain (VH) comprising complementarity determining regions CDRHI, CDRH2, and CDRH3 and a light chain variable domain (VL) comprising complementarity determining regions CDRLI, CDRL2, and CDRLS. The antigen-binding site can, for example, specifically bind a B-cell surface protein, e.g., CD19. The antigen-binding site can optionally be humanized or fully human. In certain embodiments, (i) the CDRHI, CDRH2, and CDRH3 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 CDRL3 comprise the amino acid sequences of SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively; or (ii) the CDRHI ,CDRH2, and CDRHB 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 CDRL3 comprise the amino acid sequences of SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively. In certain embodiments, (i) the Vn and the VL comprise amino acid sequences at least 95% identical to the amino acid sequences of SEQ ID NOs: 4 and 8, respectively; or (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. In certain embodiments, (i) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 4 and 8, respectively; or (ii) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 13 and 17, respectively. In certain embodiments, the antigen-binding site is present in an scFv. In certain embodiments, the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 9 and 18. In certain embodiments, the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 9 and 18. The CAR can optionally further comprise a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain. The transmembrane domain may optionally comprise 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 may optionally comprise a 4- IBB intracellular domain, e.g., wherein the 4- IBB intracellular domain comprises the amino acid sequence of SEQ ID NO: 20. The intracellular signaling domain may optionally comprise 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 optionally further comprise a hinge domain or linker 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, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 23 and 27. In certain embodiments, the CAR further comprises a killer immunoglobulin- like receptor (KIR) transmembrane domain and a KIR cytoplasmic domain.

[0035] In certain aspects, the present disclosure provides a composition of engineered T cells generated by a method provided herein. The composition can optionally further comprise a pharmaceutically acceptable carrier or excipient.

[0036] In certain aspects, the present disclosure provides a method of treating a disease in a patient in need thereof, the method comprising administering to the patient a composition provided herein. In certain embodiments, the engineered T cells are autologous to thepatient. The method can comprise, for example, administering the engineered T cells to the patient 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. The disease can be an autoimmune disease, for example, a B-cell- mediated autoimmune disease, e.g., an autoimmune disease 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, juvenile myositis, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, and immune mediated necrotizing myopathy. Alternatively, the disease can be cancer.

[0037] These and other aspects and features of the present disclosure are described in the following detailed description and claims.DESCRIPTION OF THE DRAWINGS

[0038] The invention can be more completely understood with reference to the following drawings.

[0039] FIGURE 1 depicts a schematic diagram of an exemplary method of preparing CAR T cells.

[0040] FIGURE 2A and FIGURE 2B depict schematic diagrams of exemplary anti-CD19- CAR constructs.

[0041] FIGURE 3 depicts growth curves of viable nucleated cells (VNC) during preparation of CAR T cells using a small-scale manufacturing protocol, using either whole blood (WB) or leukapheresis product (LUK) isolated from one of three healthy human donors (HD1 , HD2, HD3) as the starting material. VNC measurements were taken at Days 3, 6, and 9 of the manufacturing protocol.

[0042] FIGURE 4 depicts representative flow cytometry scatterplots showing the percentage of CD3+ cells expressing the CD19-CAR (“CAR19+ CD3 cells”) in CAR T cell compositions prepared from WB or LUK samples isolated from donor HD1. Y-axes represents the side scatter (SSC).

[0043] FIGURES 5A - 5B summarize the composition of CAR-T cell populations prepared from LUK or WB samples isolated from three healthy donors. FIGURE 5A depicts representative flow cytometry scatterplots summarizing the cellular composition of a population of live CD3+ CAR+ T cells manufactured from either a WB sample or a LUK sample isolated from donor HDL T cell memory subsets were categorized based on the expression level of CCR7 (Y-axis) and CD45RA (X-axis) relative to fluorescence minus one (FMO) controls. Tn are naive T cells; Temra are Terminal effector memory T cells; Tern are effector memory T cells; Tcm are central memory T cells. FIGURE 5B is a bar plot depicting the percentages of Tn, Temra, Tem, and Tcm cells in populations of live CD3+ CAR+ T cells prepared from WB or LUK samples isolated from three different donors.

[0044] FIGURE 6 depicts flow cytometry histograms summarizing expression of T-cell activation markers (CD25 and CD69) and T-cell exhaustion markers (PD1 and TIM3) in populations of CD3+ CD19-CAR+ cells prepared from either WB or LUK samples isolated from a healthy human donor. Results for SB and LUK samples for each marker are vertically staggered on the same plot for the sake of visualization.

[0045] FIGURE 7 depicts proliferation of CD19-CAR T cells co-cultured with (CD 19+) target Nalm6 cells at an Effector:T-cell (E:T) ratio of 1:1 (“stimulated”). CD19-CAR T cells that were not co-cultured with target cells were used as a control (“unstimulated”). CD19- CAR T cells were prepared from either a WB sample or a LUK sample isolated from the same healthy human donor, and T-cell proliferation was followed for 14 days after initial target cell stimulation. Total CAR+ cell numbers are shown as mean + SD across 3 donors; no statistical differences were observed between LUK- and WB-derived CAR-T samples at any time point.

[0046] FIGURES 8A - 8C summarize cytotoxicity of WB- and LUK-derived CD19-CAR T cells against CD 19+ Nalm6 target cells. FIGURE 8A and FIGURE 8B depict cytolysis of CD19-positive Nalm6 cells over time, measured using an imaging-based IncuCyte cytotoxicity assay during co-culture with CD19-CAR T cells at an E:T ratio of 1:1 (FIGURE 8A) or 0.125:1 (FIGURE 8B). Nalm6 cells that were not co-cultured with CAR-T cells were used as a control. Data represent mean + SD in triplicates, and the E:T ratio was calculated based on the number of transduced T-cells. FIGURE 8C is a bar graph depicting the area under the curve (AUC) for the data shown in FIGURES 8A-8B, and for all other E:T ratios tested.

[0047] FIGURES 9A and 9B depict the growth and cytotoxic activity of CD19-CAR T cells manufactured at clinical scale from either WB samples (200 mL) isolated from healthy human donors (HD4, HD5) or from LUK samples isolated from different healthy human donors (HD 6, HD7). FIGURE 9A depicts growth curves during CAR-T manufacturing, measured as the number of viable nucleated cells (VNC) at the indicated timepoints.Corresponding percentages of CAR+ cells and counts of VNCs for the clinical-scale runs are shown in TABLE 5. FIGURE 9B depicts cytolysis of CD 19+ target cells following coculture with WB-sourced CD19-CAR T cells for 120 hours at the indicated E:T ratios. Data were measured using an imaging-based IncuCyte cytotoxicity assay and quantified as AUC (mean ± SD). E:T ratios were calculated based on the number of transduced T-cells.

[0048] FIGURE 10 is a flow cytometry scatterplots showing the percentage of CD3+ cells expressing the CD19-CAR (“CAR19+ CD3 cells”) in CAR T cell compositions prepared from a WB sample (100 mL) isolated from donor having systemic lupus erythematosus (SLE). X-axis represents CD19-CAR expression; Y-axis represents the side scatter (SSC).

[0049] FIGURE 11 is a bar graph that summarizes cytolysis of (CD19+) Nalm6 target cells following co-culture with CD19-CAR T cells derived from a WB sample (100 mL) isolated from an SLE patient. Cells were co-cultured for 120 hours at the indicated E:T ratios, and data were measured using an imaging-based IncuCyte cytotoxicity assay and quantified as AUC (mean + SD). Nalm6 cells that were either not co-cultured with CAR-T cells (“Nalm6 CTRL”) or co-cultured with non-transduced T cells (“NTD”) were used as controls. E:T ratios were calculated based on the number of transduced T-cells.

[0050] FIGURES 12A - 12B summarize the composition of CAR-T cell populations prepared from 100-mL WB samples isolated from SLE patients. FIGURE 12A is a representative flow cytometry scatterplot summarizing the cellular composition of a population of live CD3+ CAR+ T cells manufactured from a WB sample isolated from a donor with SLE. T cell memory subsets were categorized based on the expression level of CCR7 (Y-axis) and CD45RA (X-axis) relative to fluorescence minus one (FMO) controls.Tn are naive T cells; Temra are Terminal effector memory T cells ; Tem are effector memory T cells; Tcm are central memory T cells. FIGURE 12B is a bar plot depicting the percentages of Tn, Temra, Tem, and Tcm cells in populations of live CD3+ CAR+ T cells prepared from 100-mL WB samples isolated from two different donors having SLE (SLE 1, SLE 2).

[0051] FIGURE 13 depicts flow cytometry histograms summarizing expression of T-cell activation markers (CD25 and CD69) and T-cell exhaustion markers (PD1 and TIM3) in populations of CD3+ CD19-CAR+ cells prepared from 100-mL WB samples isolated from different donors having SLE. Results for each donor (SLE1 and SLE2) are vertically staggered on the same plots for the sake of visualization.DETAILED DESCRIPTION

[0052] Leukapheresis is typically used to isolate immune cells from subjects for use in preparing genetically modified immune cells for adoptive cell therapy (ACT). However, access to apheresis centers presents a significant obstacle to the more widespread adoption of ACTs, such as CAR-T cell therapy. It has now been discovered that genetically modified T cells can be efficiently and effectively manufactured from primary T cells obtained from a whole blood sample isolated from a subject. Whole blood can be obtained from subjects through a simple blood draw, thereby eliminating the need for specialized apheresis centers and equipment.I. Definitions

[0053] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0054] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0055] 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.

[0056] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigenbinding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of antibodies that have been modified or engineered include chimericantibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0057] 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. et al. (1987) J. MOL. BIOL. 196:901-917). The three CDRs, referred to as CDRi, CDR2, and CDR3, contribute to the antibody binding specificity.

[0058] 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 fishes 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 et al. (1984) PROC. NATL. ACAD. SCI. U.S.A., 81: 6851-55; Morrison et al. (1985) PROC. NATL. ACAD. SCI. U.S.A., 81 :6851; Takeda et al. (1985) NATURE, 314: 452).

[0059] The term “autoantigen,” as used herein, refers to an endogenous antigen that stimulates production of an autoimmune response, such as production of autoantibodies. The term “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.

[0060] 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 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”.

[0061] 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, CD19 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 the first 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 et al. (2011) J. PHARM. BIOMED. ANAL., 54: 351-358. A CD19 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.

[0062] 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 T 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. Any of the therapeutic compositions disclosed herein may be administered to a subject in need thereof in an effective amount.

[0063] 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 the percentage 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 foraligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0064] 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.

[0065] 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. For the avoidance of doubt, as used herein, a whole blood sample is not considered to be “isolated” from a subject if the subject is undergoing or has undergone apheresis or leukapheresis.

[0066] 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, commensurate with a reasonable benefit / risk ratio.

[0067] 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. Pharmaceuticallyacceptable 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 (23d ed. 2020).

[0068] 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.

[0069] As used herein, the terms “subject” and “patient” refer to an organism to be treated by any of the methods and compositions described herein, and / or from which a blood or tissue sample (e.g., a whole blood sample) may be obtained. 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.

[0070] 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) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (c) relieving the disease, disorder, etc. , i.e., causing regression of the disease state. 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.IL Methods of Preparing Engineered T Cells

[0071] Disclosed herein, in one aspect, is a method of producing a composition of engineered T cells that express a chimeric receptor, the method comprising: (a) contacting a population of primary T cells with an activating agent, wherein the population of primary T cells has been obtained directly from a whole blood sample isolated from a subject, thereby generating a population of activated T cells; (b) introducing into the activated T cells a heterologous polynucleotide encoding the chimeric receptor, thereby generating a population of genetically modified T cells; (c) incubating the population of genetically modified T cells in a basal culture medium for at least two days, thereby generating a population of expanded T cells; and (d) harvesting the expanded T cells, thereby producing a composition of engineered T cells.

[0072] Disclosed herein, in another aspect, is method of producing a composition of engineered T cells that express a chimeric receptor, the method comprising: (a) contacting a population of primary T cells with an activating agent, wherein the population of primary T cells has been obtained from a sample isolated from a subject, thereby generating a population of activated T cells; (b) introducing into the activated T cells a heterologous polynucleotide encoding the chimeric receptor, thereby generating a population of genetically modified T cells; (c) incubating the population of genetically modified T cells in a basal culture medium for at least two days, thereby generating a population of expanded T cells; and(d) harvesting the expanded T cells, thereby producing a composition of engineered T cells, wherein the subject has an autoimmune disease, and / or the population of primary T cells is contacted with the activating agent within 72 hours of when the sample was isolated from the subject.

[0073] The foregoing methods may utilize a population of primary T cells that is or has been obtained, directly or indirectly, from a whole blood sample isolated from a subject. The whole blood sample isolated from the subject can have a volume of, for example, from 50 to 300 mL, e.g., from 50 to 275 mL, from 50 to 250 mL, from 50 to 225 mL, from 50 to 200 mL, from 50 to 175 mL, from 50 to 150 mL, from 50 to 125 mL, from 50 to 100 mL, from 50 to 75 mL, from 75 to 300 mL, from 75 to 275 mL, from 75 to 250 mL, from 75 to 225 mL, from 75 to 200 mL, from 75 to 175 mL, from 75 to 150 mL, from 75 to 125 mL, from 75 to 100 mL, from 100 to 300 mL, from 100 to 275 mL, from 100 to 250 mL, from 100 to 225 mL, from 100 to 200 mL, from 100 to 175 mL, from 100 to 150 mL, from 100 to 125 mL, from 125 to 300 mL, from 125 to 275 mL, from 125 to 250 mL, from 125 to 225 mL, from125 to 200 mL, from 125 to 175 mL, from 125 to 150 mL, from 150 to 300 mL, from 150 to 275 mL, from 150 to 250 mL, from 150 to 225 mL, from 150 to 200 mL, from 150 to 175 mL, from 175 to 300 mL, from 175 to 275 mL, from 175 to 250 mL, from 175 to 225 mL, from 175 to 200 mL, from 200 to 300 mL, from 200 to 275 mL, from 200 to 250 mL, from 200 to 225 mL, from 225 to 300 mL, from 225 to 275 mL, from 225 to 250 mL, from 250 to 300 mL, from 250 to 275 mL, or from 275 to 300 mL. In certain embodiments, the volume of the whole blood sample isolated from the subject is from 100 to 300 or from 100 to 200 mL. In certain embodiments, the volume of the whole blood sample isolated from the subject is no greater than 300 mL, e.g., no greater than 275 mL, no greater than 250 mL, no greater than 225 mL, no greater than 200 mL, no greater than 175 mL, no greater than 150 mL, no greater than 125 mL, no greater than 100 mL, no greater than 75 mL, or no greater than 50 mL. In certain embodiments, the volume of the whole blood sample isolated from the subject is no greater than 200 mL.

[0074] In certain embodiments, the sample (e.g. whole blood sample) obtained from the subject is never subjected to apheresis or leukapheresis.

[0075] In certain embodiments, the sample (e.g., whole blood sample) is or has been isolated from a subject having an autoimmune disease, e.g., an autoimmune disease to be treated using the compositions and methods disclosed herein. The autoimmune disease can be a B-cell mediated autoimmune disease, e.g., an autoimmune disease selected from: 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, 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, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, systemic sclerosis with renal involvement, systemic sclerosis with pulmonary involvement, systemic sclerosis with cardiac involvement, 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, the subject has an autoimmune disease selected from SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV,mucocutaneous PV, MG, MuSK-associated MG, AChR MG, myositis, juvenile myositis, systemic sclerosis, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, systemic sclerosis with renal involvement, systemic sclerosis with pulmonary involvement, systemic sclerosis with cardiac involvement, chronic immune demyelinating polyneuropathy, membranous nephropathy, anti- synthetase syndrome, dermatomyositis, and immune mediated necrotizing myopathy. In certain embodiments, the subject has an autoimmune disease selected from SLE, PV, MG, myositis, systemic sclerosis, multiple sclerosis, membranous nephropathy, and chronic immune demyelinating polyneuropathy. In certain embodiments, the subject has an autoimmune disease selected from SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK-associated MG, AChR MG, myositis, juvenile myositis, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, systemic sclerosis with renal involvement, systemic sclerosis with pulmonary involvement, systemic sclerosis with cardiac involvement, membranous nephropathy, anti-synthetase syndrome, dermatomyositis, and immune mediated necrotizing myopathy. In certain embodiments, the subject has SLE. a. T-cell Activation

[0076] In certain embodiments, the methods of preparing engineered T cells disclosed herein comprise a step of contacting a population of primary T cells with an activating agent, thereby generating a population of activated T cells. This step may be referred to herein as “step (a).” This contacting may activate or stimulate T cells (e.g., CD4+ and / or CD8+ T cells) via the activation or stimulation of CD3 / TCR complex signaling, and / or via the activation or stimulation of a costimulatory molecule and / or growth factor receptor on the surface of the cells. In certain embodiments, the activating agent comprises an antibody or an antigen-binding fragment thereof.

[0077] In certain embodiments, the activating agent comprises an agent that activates or stimulates CD3 / TCR complex signaling. For example, in certain embodiments, the agent that activates or stimulates CD3 / TCR complex signaling is selected from a small molecule, a ligand, or an antibody, e.g., an anti-CD3 antibody, such as a humanized or fully human anti- CD3 antibody. In certain embodiments, the activating agent comprises an agent that activates or stimulates costimulatory signaling, e.g., CD28, ICOS, CD27, CD25, 4-1BB, 0X40, HVEM, LIGHT, CD40, DR3, GITR, CD30, TIM1, CD2, CD226, IL6RA, and / or IL6RB signaling. For example, in certain embodiments, the agent that activates or stimulatescostimulatory signaling is selected from a small molecule, a ligand, or an antibody, e.g., an antibody or antigen-binding fragment thereof that specifically binds one of the foregoing proteins. In certain embodiments, the activating agent comprises an agent that activates or stimulates CD28 signaling, e.g., an anti-CD28 antibody, such as a humanized or fully human anti-CD28 antibody or antigen-binding fragment thereof. In certain embodiments, the activating agent comprises an anti-CD3 antibody and an anti-CD28 antibody. In certain embodiments, the activating agent comprises a multispecific antibody, e.g., an antibody that specifically binds CD3 and CD28. In certain embodiments, the activating agent comprises a humanized anti-CD3 antibody and a humanized anti-CD28 antibody.

[0078] In certain embodiments wherein the activating agent comprises an anti-CD3 antibody and an anti-CD28 antibody, the ratio of anti-CD3:anti-CD28 antibody ranges from 100:1 to 1: 100. In certain embodiments, the activating agent comprises more anti-CD28 antibody than anti-CD3 antibody, i.e., the ratio of anti-CD3:anti-CD28 is less than one. In other embodiments, the activating agent comprises more anti-CD3 antibody than anti-CD28 antibody, i.e., the ratio of anti-CD3:anti-CD28 is greater than one. In certain embodiments, the activating agent comprises approximately equal amounts of anti-CD3 antibody and anti- CD28, i.e., the ratio of anti-CD3:anti-CD28 antibody is approximately 1. In certain embodiments, the activating agent comprises anti-CD3 and anti-CD28 antibody at a ratio of from 100:1 to 1: 100, e.g., from 100: 1 to 1 :75, from 100:1 to 1:50, from 100:1 to 1:30, from 100:1 to 1:20, from 100:1 to 1 :10, from 100:1 to 1:5, from 100:1 to 1:3, from 100: 1 to 1 :2, from 100:1 to 1: 1, from 100:1 to 2:1, from 100: 1 to 3:1, from 100: 1 to 5: 1, from 100:1 to 10: 1, from 100:1 to 20:1 , from 100:1 to 30:1, from 100: 1 to 50: 1, from 100:1 to 75:1, from 75: 1 to 1 :100, from 75:1 to 1:75, from 75:1 to 1 :50, from 75:1 to 1:30, from 75:1 to 1:20, from 75:1 to 1 :10, from 75:1 to 1:5, from 75: 1 to 1 :3, from 75:1 to 1:2, from 75:1 to 1 :1, from 75: 1 to 2:1, from 75:1 to 3:1, from 75:1 to 5:1, from 75:1 to 10:1, from 75: 1 to 20: 1, from 75: 1 to 30: 1, from 75: 1 to 50: 1, from 50: 1 to 1:100, from 50: 1 to 1 :75, from 50: 1 to 1 :50, from 50:1 to 1 :30, from 50:1 to 1 :20, from 50:1 to 1 :10, from 50: 1 to 1 :5, from 50: 1 to 1 :3, from 50:1 to 1 :2, from 50:1 to 1 :1, from 50:1 to 2:1, from 50:1 to 3:1, from 50:1 to 5:1, from 50: 1 to 10: 1, from 50:1 to 20: 1, from 50:1 to 30:1, from 30:1 to 1:100, from 30:1 to 1:75, from 30: 1 to 1 :50, from 30: 1 to 1:30, from 30: 1 to 1:20, from 30:1 to 1: 10, from 30: 1 to 1:5, from 30: 1 to 1 :3, from 30: 1 to 1 :2, from 30: 1 to 1:1, from 30: 1 to 2: 1 , from 30: 1 to 3: 1 , from 30: 1 to 5:1, from 30:1 to 10: 1, from 30:1 to 20:1, from 20: 1 to 1 :100, from 20:1 to 1:75, from 20: 1 to 1 :50, from 20:1 to 1 :30, from 20:1 to 1:20, from 20:1 to 1:10, from 20:1 to 1:5, from20:1 to 1:3, from 20:1 to 1:2, from 20:1 to 1:1, from 20:1 to 2:1, from 20:1 to 3:1, from 20:1 to 5:1, from 20:1 to 10:1, from 10:1 to 1:100, from 10:1 to 1:75, from 10:1 to 1:50, from 10:1 to 1:30, from 10:1 to 1:20, from 10:1 to 1:10, from 10:1 to 1:5, from 10:1 to 1:3, from 10:1 to 1:2, from 10:1 to 1:1, from 10:1 to 2:1, from 10:1 to 3:1, from 10:1 to 5:1, from 5:1 to 1:100, from 5:1 to 1:75, from 5:1 to 1:50, from 5:1 to 1:30, from 5:1 to 1:20, from 5:1 to 1:10, from 5:1 to 1:5, from 5:1 to 1:3, from 5:1 to 1:2, from 5:1 to 1:1, from 5:1 to 2:1, from 5:1 to 3:1, from 3:1 to 1:100, from 3:1 to 1:75, from 3:1 to 1:50, from 3:1 to 1:30, from 3:1 to 1:20, from 3:1 to 1:10, from 3:1 to 1:5, from 3:1 to 1:3, from 3:1 to 1:2, from 3:1 to 1:1, from 3:1 to 2:1, from 2:1 to 1:100, from 2:1 to 1:75, from 2:1 to 1:50, from 2:1 to 1:30, from 2:1 to 1:20, from 2:1 to 1:10, from 2:1 to 1:5, from 2:1 to 1:3, from 2:1 to 1:2, from 2:1 to 1:1, from 1:1 to 1:100, from 1:1 to 1:75, from 1:1 to 1:50, from 1:1 to 1:30, from 1:1 to 1:20, from 1:1 to 1:10, from 1:1 to 1:5, from 1:1 to 1:3, from 1:1 to 1:2, from 1:2 to 1:100, from 1:2 to 1:75, from 1:2 to 1:50, from 1:2 to 1:30, from 1:2 to 1:20, from 1:2 to 1:10, from 1:2 to 1:5, from 1:2 to 1:3, from 1:3 to 1:100, from 1:3 to 1:75, from 1:3 to 1:50, from 1:3 to 1:30, from 1:3 to 1:20, from 1:3 to 1:10, from 1:3 to 1:5, from 1:5 to 1:100, from 1:5 to 1:75, from 1:5 to 1:50, from 1:5 to 1:30, from 1:5 to 1:20, from 1:5 to 1:10, from 1:10 to 1:100, from 1:10 to 1:75, from 1:10 to 1:50, from 1:10 to 1:30, from 1:10 to 1:20, from 1:20 to 1:100, from 1:20 to 1:75, from 1:20 to 1:50, from 1:20 to 1:30, from 1:30 to 1:100, from 1:30 to 1:75, from 1:30 to 1:50, from 1:50 to 1:100, from 1:50 to 1:75, or from 1:75 to 1:100.

[0079] In certain embodiments, the activating agent (e.g., an agent comprising an anti-CD3 antibody and an anti-CD28 antibody) comprises and / or is covalently bound to a polymeric nanomatrix, e.g., a colloidal polymeric nanomatrix. For example, in certain embodiments, the activating agent comprises, or is, T Cell TransAct™. In embodiments wherein the activating agent comprises an anti-CD3 antibody and an anti-CD28 antibody, each antibody may be bound to the same polymeric nanomatrix, or to separate polymeric nanomatrices. In certain embodiments, the activating agent is comprised in a polymeric matrix comprising, e.g., biodegradable or biocompatible inert material, which can be non-toxic to cells. In certain embodiments, the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains. In certain embodiments, the mobile matrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. A polysaccharide may include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum, or alginate. Other polymersmay include polyesters, polyethers, poly acrylates, polyacrylamides, polyamines, polyethylene imines, polyquatemium polymers, polyphosphazenes, polyvinylalcohols, polyvinylacetates, polyvinylpyrrolidones, block copolymers, or polyurethanes.

[0080] In certain embodiments wherein the activating agent comprises and / or is covalently bound to a polymeric nanomatrix (e.g., a colloidal polymeric nanomatrix), the nanomatrix may be from 1 nm to 500 nm in size, e.g., from 1 nm to 400 nm, from 1 nm to 300 nm, from 1 nm to 200 nm, from 1 nm to 100 nm, from 1 nm to 50 nm, from 1 nm to 25 nm, from 1 nm to 10 nm, from 10 nm to 500 nm, from 10 nm to 400 nm, from 10 nm to 300 nm, from 10 nm to 200 nm, from 10 nm to 100 nm, from 10 nm to 50 nm, from 10 nm to 25 nm, from 25 nm to 500 nm, from 25 nm to 400 nm, from 25 nm to 300 nm, from 25 nm to 200 nm, from 25 nm to 100 nm, from 25 nm to 50 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to 300 nm, from 50 nm to 200 nm, from 50 nm to 100 nm, from 100 nm to 500 nm, from 100 nm to 400 nm, from 100 nm to 300 nm, from 100 nm to 200 nm, from 200 nm to 500 nm, from 200 nm to 400 nm, from 200 nm to 300 nm, from 300 nm to 500 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm in size. In certain embodiments, the nanomatrix is from 10 nm to 200 nm in size.

[0081] In certain embodiments, the activating agent comprises and / or is covalently bound to a particle, e.g., a bead. In certain embodiments, the bead is biocompatible, i.e., composed of a material that is suitable for biological use. In certain embodiments, the beads are nontoxic to cultured cells, e.g., cultured T cells. In some embodiments, the bead has a diameter of 0.1 pm to 10 pm, e.g. 0.1 pm to 7.5 pm, 0.1 pm to 5 pm, 0.1 pm to 4 pm, 0.1 pm to 3 pm, 0.1 pm to 2 pm, 0.1 pm to 1 pm, 0.1 pm to 0.5 pm, 0.5 pm to 10 pm, 0.5 pm to 7.5 pm, 0.5 pm to 5 pm, 0.5 pm to 4 pm, 0.5 pm to 3 pm, 0.5 pm to 2 pm, 0.5 pm to 1 pm, 1 pm to 10 pm, 1 pm to 7.5 pm, 1 pm to 5 pm, 1 pm to 4 pm, 1 pm to 3 pm, 1 pm to 2 pm, 2 pm to 10 pm, 2 pm to 7.5 pm, 2 pm to 5 pm, 2 pm to 4 pm, 2 pm to 3 pm, 3 pm to 10 pm, 3 pm to 7.5 pm, 3 pm to 5 pm, 3 pm to 4 pm, 4 pm to 10 pm, 4 pm to 7.5 pm, 4 pm to 5 pm, 5 pm to 10 pm, 5 pm to 7.5 pm, or 7.5 pm to 10 pm. In certain embodiments, the bead has a diameter of 1 pm to 5 pm.

[0082] In certain embodiments, the bead is a magnetic bead. In particular embodiments, the bead comprises a magnetic core e.g., DynaBeads® or Miltenyi ExpACT® beads). In certain embodiments, the magnetic core comprises a metal, optionally selected from iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium or any combinations thereof. In certain embodiments, the magnetic core comprises metal oxides(e.g., iron oxides), ferrites (e.g., manganese ferrites, cobalt ferrites, nickel ferrites, etc.), hematite, and / or metal alloys. In certain embodiments, the magnetic core comprises one or more of a ferrite, a metal, a metal alloy, an iron oxide, or chromium dioxide. In certain embodiments, the inner core comprises an iron oxide e.g., FeaC ). The bead containing a magnetic core may optionally be covered by a surface functionalized coat or coating. In certain embodiments, the coat can contain a material that can include, e.g., a polymer, a polysaccharide, a silica, a fatty acid, a protein, a carbon, agarose, sepharose, or a combination thereof. In certain embodiments, the polymer is a polyethylene glycol, poly (lactic-co- glycolic acid), poly glutaraldehyde, polyurethane, polystyrene, or a polyvinyl alcohol. In certain embodiments, the outer coat or coating comprises polystyrene. In particular embodiments, the outer coating is surface functionalized.

[0083] In some embodiments, the stimulatory reagent comprises a bead that contains a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coat comprises at least one polysaccharide (e.g., amino dextran), at least one polymer (e.g., polyurethane), and silica. In certain embodiments, the metal oxide core is a colloidal iron oxide core.

[0084] In certain embodiments, an activating agent is covalently bound to a bead as described herein, and the population of primary T cells is incubated with the beads bound to the activating agent at a bead:cell ratio from 3:1 to 0.2: 1, e.g., from 2.5: 1 to 0.2: 1, from 2: 1 to 0.2:1 , from 1.5:1 to 0.2:1, from 1.25: 1 to 0.2:1, from 1 :1 to 0.2: 1, from 0.75:1 to 0.2: 1, from 0.5:1 to 0.2:1, from 3:1 to 0.5:1, from 2.5:1 to 0.5:1, from 2:1 to 0.5:1, from 1.5: 1 to 0.5:1, from 1.25: 1 to 0.5: 1, from 1:1 to 0.5:1, from 0.75:1 to 0.5:1, from 3:1 to 0.75: 1, from 2.5: 1 to 0.75:1, from 2: 1 to 0.75:1, from 1.5:1 to 0.75:1, from 1.25: 1 to 0.75: 1, from 1: 1 to 0.75: 1, from 3:1 to 1: 1, from 2.5: 1 to 1 :1, from 2: 1 to 1 :1, from 1.5:1 to 1 :1, from 1.25:1 to 1 :1, from 3:1 to 1.25:1, from 2.5:1 to 1.25:1, from 2:1 to 1.25:1, from 1.5:1 to 1.25:1, from 3:1 to 1.5: 1, from 2.5:1 to 1.5:1, from 2:1 to 1.5:1, from 3: 1 to 2:1, from 2.5: 1 to 2:1, or from 3:1 to 2.5:1. In certain embodiments, the population of primary T cells is incubated with the beads at a bead:cell ratio of at least 0.2: 1 , at least 0.5: 1 , at least 0.75: 1 , at least 1 : 1 , at least 1.25: 1 , at least 1.5: 1, at least 1.75:1, at least 2:1, at least 2.5:1, or at least 3: 1.

[0085] In certain embodiments, the activating agent does not comprise a bead, and / or the activating agent is not covalently bound to a bead.

[0086] In certain embodiments, the population of primary T cells is contacted with the activating agent for 0.5 to 48 hours. For example, in certain embodiments, step (a) comprises contacting or incubating the population of primary T cells with the activating agent for 0.5 to 48 hours, 0.5 to 40 hours, 0.5 to 36 hours, 0.5 to 32 hours, 0.5 to 28 hours, 0.5 to 24 hours, 0.5 to 20 hours, 0.5 to 18 hours, 0.5 to 16 hours, 0.5 to 14 hours, 0.5 to 12 hours, 0.5 to 10 hours, 0.5 to 8 hours, 0.5 to 7 hours, 0.5 to 6 hours, 0.5 to 5 hours, 0.5 to 4 hours, 0.5 to 3 hours, 0.5 to 2 hours, 0.5 to 1 hours, 1 to 48 hours, 1 to 40 hours, 1 to 36 hours, 1 to 32 hours, 1 to 28 hours, 1 to 24 hours, 1 to 20 hours, 1 to 18 hours, 1 to 16 hours, 1 to 14 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, 2 to 48 hours, 2 to 40 hours, 2 to 36 hours, 2 to 32 hours, 2 to 28 hours, 2 to 24 hours, 2 to 20 hours, 2 to 18 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 7 hours, 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 48 hours, 3 to 40 hours, 3 to 36 hours, 3 to 32 hours, 3 to 28 hours, 3 to 24 hours, 3 to 20 hours, 3 to 18 hours, 3 to 16 hours, 3 to 14 hours, 3 to 12 hours, 3 to 10 hours, 3 to 8 hours, 3 to 7 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 48 hours, 4 to 40 hours, 4 to 36 hours, 4 to 32 hours, 4 to 28 hours, 4 to 24 hours, 4 to 20 hours, 4 to 18 hours, 4 to 16 hours, 4 to 14 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 7 hours, 4 to 6 hours, 4 to 5 hours, 5 to 48 hours, 5 to 40 hours, 5 to 36 hours, 5 to 32 hours, 5 to 28 hours, 5 to 24 hours, 5 to 20 hours, 5 to 18 hours, 5 to 16 hours, 5 to 14 hours, 5 to 12 hours, 5 to 10 hours, 5 to 8 hours, 5 to 7 hours, 5 to 6 hours, 6 to 48 hours, 6 to 40 hours, 6 to 36 hours, 6 to 32 hours, 6 to 28 hours, 6 to 24 hours, 6 to 20 hours, 6 to 18 hours, 6 to 16 hours, 6 to 14 hours, 6 to 12 hours,6 to 10 hours, 6 to 8 hours, 6 to 7 hours, 7 to 48 hours, 7 to 40 hours, 7 to 36 hours, 7 to 32 hours, 7 to 28 hours, 7 to 24 hours, 7 to 20 hours, 7 to 18 hours, 7 to 16 hours, 7 to 14 hours,7 to 12 hours, 7 to 10 hours, 7 to 8 hours, 8 to 48 hours, 8 to 40 hours, 8 to 36 hours, 8 to 32 hours, 8 to 28 hours, 8 to 24 hours, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours,8 to 12 hours, 8 to 10 hours, 10 to 48 hours, 10 to 40 hours, 10 to 36 hours, 10 to 32 hours, 10 to 28 hours, 10 to 24 hours, 10 to 20 hours, 10 to 18 hours, 10 to 16 hours, 10 to 14 hours, 10 to 12 hours, 12 to 48 hours, 12 to 40 hours, 12 to 36 hours, 12 to 32 hours, 12 to 28 hours, 12 to 24 hours, 12 to 20 hours, 12 to 18 hours, 12 to 16 hours, 12 to 14 hours, 14 to 48 hours, 14 to 40 hours, 14 to 36 hours, 14 to 32 hours, 14 to 28 hours, 14 to 24 hours, 14 to 20 hours, 14 to 18 hours, 14 to 16 hours, 16 to 48 hours, 16 to 40 hours, 16 to 36 hours, 16 to 32 hours, 16 to 28 hours, 16 to 24 hours, 16 to 20 hours, 16 to 18 hours, 18 to 48 hours, 18 to 40 hours, 18 to 36 hours, 18 to 32 hours, 18 to 28 hours, 18 to 24 hours, 18 to 20 hours, 20 to 48 hours, 20 to 40 hours, 20 to 36 hours, 20 to 32 hours, 20 to 28 hours, 20 to 24 hours, 24 to 48 hours, 24to 40 hours, 24 to 36 hours, 24 to 32 hours, 24 to 28 hours, 28 to 48 hours, 28 to 40 hours, 28 to 36 hours, 28 to 32 hours, 32 to 48 hours, 32 to 40 hours, 32 to 36 hours, 36 to 48 hours, 36 to 40 hours, or 40 to 48 hours. In certain embodiments, step (a) comprises contacting or incubating the population of primary T cells with the activating agent for at least 0.5, 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 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 24, at least 28, at least 32, at least 36, at least 40, or at least 48 hours. In certain embodiments, step (a) comprises contacting or incubating the population of primary T cells with the activating agent for at least 12 hours or at least 24 hours.

[0087] In certain embodiments, the step of contacting the population of primary T cells is carried out in a basal culture medium. The basal culture medium may comprise, for example, TexMACS™ medium (Miltenyi Biotec) or OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher). In certain embodiments, the basal culture medium does not comprise serum. In certain embodiments, the basal culture medium is supplemented with one or more additional components for the maintenance, expansion, and / or activation of T cells (e.g., through supplementation with OpTmizer™ T-Cell Expansion Supplement (ThermoFisher)). In certain embodiments, the basal culture medium further comprises a serum replacement supplement, for example, an immune cell serum replacement, e.g., ThermoFisher, #A2596101, the CTS™ Immune Cell Serum Replacement, or the immune cell serum replacement described in Smith et al. (2015) CLIN. TRANSL. IMMUNOLOGY, 4(1): e31. In certain embodiments, the basal culture medium comprises a free form of an amino acid, such as L-glutamine. In certain embodiments, the basal culture medium comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher).

[0088] In certain embodiments, the basal culture medium used in step (a) comprises one or more cytokines, e.g., recombinant cytokines. For example, in certain embodiments, the basal culture medium used in step (a) comprises wild-type or recombinant human IL-2, wild-type or recombinant human IL-7, and / or wild-type or recombinant human IL- 15. In certain embodiments, the basal culture medium used in step (a) comprises human IL-7 (e.g., wildtype or recombinant IL-7) and human IL-15 (e.g., wild-type or recombinant IL-15). In certain embodiments, the basal culture medium used in step (a) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild-type or recombinant IL- 15) at a concentration of 1 to 100 ng / mL, e.g., 1 to 90 ng / mL, 1 to 80 ng / mL, 1 to 70 ng / mL, 1 to 60ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 35 ng / mL, 1 to 30 ng / mL, 1 to 25 ng / mL, 1 to 20 ng / mL, 1 to 15 ng / mL, 1 to 12.5 ng / mL, 1 to 10 ng / mL, 1 to 7.5 ng / mL, 1 to 5 ng / mL, 5 to 100 ng / mL, 5 to 90 ng / mL, 5 to 80 ng / mL, 5 to 70 ng / mL, 5 to 60 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 5 to 35 ng / mL, 5 to 30 ng / mL, 5 to 25 ng / mL, 5 to 20 ng / mL, 5 to 15 ng / mL, 5 to 12.5 ng / mL, 5 to 10 ng / mL, 5 to 7.5 ng / mL, 7.5 to 100 ng / mL, 7.5 to 90 ng / mL, 7.5 to 80 ng / mL, 7.5 to 70 ng / mL, 7.5 to 60 ng / mL, 7.5 to 50 ng / mL, 7.5 to 40 ng / mL, 7.5 to 35 ng / mL, 7.5 to 30 ng / mL, 7.5 to 25 ng / mL, 7.5 to 20 ng / mL, 7.5 to 15 ng / mL, 7.5 to 12.5 ng / mL, 7.5 to 10 ng / mL, 10 to 100 ng / mL, 10 to 90 ng / mL, 10 to 80 ng / mL, 10 to 70 ng / mL,10 to 60 ng / mL, 10 to 50 ng / mL, 10 to 40 ng / mL, 10 to 35 ng / mL, 10 to 30 ng / mL, 10 to 25 ng / mL, 10 to 20 ng / mL, 10 to 15 ng / mL, 10 to 12.5 ng / mL, 12.5 to 100 ng / mL, 12.5 to 90 ng / mL, 12.5 to 80 ng / mL, 12.5 to 70 ng / mL, 12.5 to 60 ng / mL, 12.5 to 50 ng / mL, 12.5 to 40 ng / mL, 12.5 to 35 ng / mL, 12.5 to 30 ng / mL, 12.5 to 25 ng / mL, 12.5 to 20 ng / mL, 12.5 to 15 ng / mL, 15 to 100 ng / mL, 15 to 90 ng / mL, 15 to 80 ng / mL, 15 to 70 ng / mL, 15 to 60 ng / mL, 15 to 50 ng / mL, 15 to 40 ng / mL, 15 to 35 ng / mL, 15 to 30 ng / mL, 15 to 25 ng / mL, 15 to 20 ng / mL, 20 to 100 ng / mL, 20 to 90 ng / mL, 20 to 80 ng / mL, 20 to 70 ng / mL, 20 to 60 ng / mL, 20 to 50 ng / mL, 20 to 40 ng / mL, 20 to 35 ng / mL, 20 to 30 ng / mL, 20 to 25 ng / mL, 25 to 100 ng / mL, 25 to 90 ng / mL, 25 to 80 ng / mL, 25 to 70 ng / mL, 25 to 60 ng / mL, 25 to 50 ng / mL, 25 to 40 ng / mL, 25 to 35 ng / mL, 25 to 30 ng / mL, 30 to 100 ng / mL, 30 to 90 ng / mL, 30 to 80 ng / mL, 30 to 70 ng / mL, 30 to 60 ng / mL, 30 to 50 ng / mL, 30 to 40 ng / mL, 30 to 35 ng / mL, 35 to 100 ng / mL, 35 to 90 ng / mL, 35 to 80 ng / mL, 35 to 70 ng / mL, 35 to 60 ng / mL, 35 to 50 ng / mL, 35 to 40 ng / mL, 40 to 100 ng / mL, 40 to 90 ng / mL, 40 to 80 ng / mL, 40 to 70 ng / mL, 40 to 60 ng / mL, 40 to 50 ng / mL, 50 to 100 ng / mL, 50 to 90 ng / mL, 50 to 80 ng / mL, 50 to 70 ng / mL, 50 to 60 ng / mL, 60 to 100 ng / mL, 60 to 90 ng / mL, 60 to 80 ng / mL, 60 to 70 ng / mL, 70 to 100 ng / mL, 70 to 90 ng / mL, 70 to 80 ng / mL, 80 to 100 ng / mL, 80 to 90 ng / mL, or 90 to 100 ng / mL. In certain embodiments, the basal culture medium used in step (a) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild-type or recombinant IL-15) at a concentration of 10 to 15 ng / mL, e.g., at a concentration of 12.5 ng / mL. In certain embodiments, the basal culture medium used in step (a) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL-15 (e.g., wild-type or recombinant IL- 15) at a concentration of at least 1 ng / mL, at least 5 ng / mL, at least 10 ng / mL, at least 12.5 ng / mL, at least 15 ng / mL, at least 20 ng / mL, at least 25 ng / mL, at least 30 ng / mL, at least 35 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, or at least 100 ng / mL. In certain embodiments, the basal culture medium used in step (a) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild- type or recombinant IL- 15) at a concentration of at least 10 ng / mL or at least 12.5 ng / mL.

[0089] In certain embodiments, step (a) comprises seeding the population of primary T cells in the basal culture medium. In certain embodiments, the population of primary T cells is seeded in the basal culture medium at a concentration of 5 x 105to 5 x 106cells / mL, e.g., at a concentration of 5 x 105to 4xl06cells / mL, 5 x 105to 3 x 106cells / mL, 5 x 105to 2 x 106cells / mL, 5 x 105to 1 x 106cells / mL, 5 x 105to 9 x 105cells / mL, 5 x 105to 8 x 105cells / mL, 5 x 105to 7 x 105cells / mL, 5 x 105to 6 x 105cells / mL, 6 x 105to 5 x 106cells / mL, 6 x 105to4 x 106cells / mL, 6 x 105to 3 x 106cells / mL, 6 x 105to 2 x 106cells / mL, 6 x 105to 1 x 106cells / mL, 6 x 105to 9 x 105cells / mL, 6 x 105to 8 x 105cells / mL, 6 x 105to 7 x 105cells / mL,7 x 105to 5 x 106cells / mL, 7 x 105to 4 x 106cells / mL, 7 x 105to 3 x 106cells / mL, 7 x 105to2 x 106cells / mL, 7 x 105to 1 x 106cells / mL, 7 x 105to 9 x 105cells / mL, 7 x 105to 8 x 105cells / mL, 8 x 105to 5 x 106cells / mL, 8 x 105to 4 x 106cells / mL, 8 x 105to 3 x 106cells / mL,8 x 105to 2 x 106cells / mL, 8 x 105to 1 x 106cells / mL, 8 x 105to 9 x 105cells / mL, 9 x 105to5 x 106cells / mL, 9 x 105to 4 x 106cells / mL, 9 x 105to 3 x 106cells / mL, 9 x 105to 2 x 106cells / mL, 9 x 10 to 1 x 106cells / mL, 1 x 106to 5 x 106cells / mL, 1 x 106to 4 x 106cells / mL, 1 x 106to 3 x 106cells / mL, 1 x 106to 2 x 106cells / mL, 2 x 106to 5 x 106cells / mL, 2 x 106to4 x 106cells / mL, 2 x 106to 3 x 106cells / mL, 3 x 106to 5 x 106cells / mL, 3 x 106to 4 x 106cells / mL, or 4 x 106to 5 x 106cells / mL. In certain embodiments, the population of primary T cells is seeded in the basal culture medium at a concentration of at least 5 x 105cells / mL, e.g., at least 6 x 105cells / mL, at least 7 x 105cells / mL, at least 8 x 105cells / mL, at least 9 x 105cells / mL, at least 1 x 106cells / mL, at least 2 x 106cells / mL, at least 3 x 106cells / mL, at least 4 x 106cells / mL, or at least 5 x 106cells / mL. In certain embodiments, the population of primary T cells is seeded in the basal culture medium at a concentration of no more than 5 x 106cells / mL, e.g., no more than 4 x 106cells / mL, no more than 3 x 106cells / mL, no more than 2 x 106cells / mL, no more than 1 x 106cells / mL, no more than 9 x 105cells / mL, no more than 8 x 105cells / mL, no more than 7 x 105cells / mL, no more than 6 x 105cells / mL, or no more than 5 x 105cells / mL. The population of primary T cells can be seeded in a volume of the basal culture medium ranging from, for example, 50 mL to 300 mL, e.g., 50 mL to 250 mL, 50 mL to 200 mL, 50 mL to 150 mL, 50 mL to 100 mL, 50 mL to 70 mL, 70 mL to 300 mL, 70 mL to 250 mL, 70 mL to 200 mL, 70 mL to 150 mL, 70 mL to 100 mL, 100 mL to 300 mL, 100 mL to 250 mL, 100 mL to 200 mL, 100 mL to 150 mL, 150 mL to 300 mL, 150 mL to 250 mL, 150 mL to 200 mL, 200 mL to 300 mL, 200 mL to 250 mL, or 250 mL to 300mL. In certain embodiments, the population of primary T cells is seeded in 50 mL to 100 mL of basal culture medium, e.g., in 70 mL of basal culture medium.

[0090] In certain embodiments, the activating agent (e.g., comprising a polymeric nanomatrix comprising anti-CD3 and anti-CD28 antibodies) is present in the basal culture medium before the population of primary T cells is added to the medium. In other embodiments, the activating agent (e.g., comprising a polymeric nanomatrix comprising anti- CD3 and anti-CD28 antibodies) is added to the basal culture medium simultaneously with the population of primary T cells, or the activating agent is added to basal culture medium that has already been seeded with the population of primary T cells.

[0091] In certain embodiments, the activating agent comprises a polymeric nanomatrix (e.g., comprising anti-CD3 and anti-CD28 antibodies), and the activating agent is used at a concentration (measured as the ratio of volume of polymeric matrix : volume of basal culture medium) of 1 :70 to 10:70, e.g., 1 :70 to 8:70, 1:70 to 6:70, 1:70 to 5:70, 1:70 to 4:70, 1:70 to 3:70, 1:70 to 2:70, 2:70 to 10:70, 2:70 to 8:70, 2:70 to 6:70, 2:70 to 5:70, 2:70 to 4:70, 2:70 to 3:70, 3:70 to 10:70, 3:70 to 8:70, 3:70 to 6:70, 3:70 to 5:70, 3:70 to 4:70, 4:70 to 10:70, 4:70 to 8:70, 4:70 to 6:70, 4:70 to 5:70, 5:70 to 10:70, 5:70 to 8:70, 5:70 to 6:70, 6:70 to 10:70, 6:70 to 8:70, or 8:70 to 10:70. In certain embodiments, the polymeric nanomatrix activating agent is used at a concentration (measured as the ratio of volume of matrix : volume of basal culture medium) of from 3:70 to 5:70, e.g., 4:70.

[0092] In certain embodiments, the population of primary T cells is contacted with the activating agent within some period of time from when the sample (e.g., whole blood sample) is isolated from the subject. For example, in certain embodiments, the population of primary T cells is contacted with the activating agent within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day of when the sample (e.g., whole blood sample) is isolated from the subject. In certain embodiments, the population of primary T cells is contacted with the activating agent within 72 hours, within 48 hours, or within 24 hours of when the sample (e.g., whole blood sample) is isolated from the subject.

[0093] T-cell activation can optionally be carried out in any suitable cell processing system known in the art, including, but not limited to, the CliniMACS Prodigy™ system (Miltenyi Biotec), or the Dynacellect™ system (Thermo Fisher). In certain embodiments, T-cell activation is carried out carried out in a suitable bioreactor known in the art, including, but not limited to, a bioreactor comprising an oxygen-permeable membrane (e.g., a G-Rex™ bioreactor).

[0094] In certain embodiments, the population of primary T cells contacted with the activating agent comprises CD8+ T cells. In certain embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the population of primary T cells are CD8+ T cells. In certain embodiments, the population of primary T cells contacted with the activating agent 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 population of primary T cells are CD4+ T cells. In certain embodiments, the population of primary T cells comprises CD4+ T cells and CD8+ T cells at a CD4:CD8 T cell ratio of from 1:5 to 5: 1, e.g., from 1 :5 to 5:1, from 1 :5 to 4:1, from 1 :5 to 3:1, from 1:5 to 2:1, from 1:5 to 1: 1, from 1:5 to 1:2, from 1:5 to 1:3, from 1:5 to 1:4, from 1:4 to 5:1, from 1:4 to 4:1, from 1:4 to 3: 1, from 1:4 to 2: 1, from 1:4 to 1: 1, from 1:4 to 1:2, from 1:4 to 1 :3, from 1:3 to 5:1 , from 1 :3 to 4: 1 , from 1 :3 to 3: 1 , from 1 :3 to 2: 1 , from 1 :3 to 1: 1, from 1 :3 to 1 :2, from 1:2 to 5: 1, from 1:2 to 4:1, from 1:2 to 3:1, from 1:2 to 2: 1, from 1:2 to 1:1, from 1:1 to 5:1, from 1:1 to 4: 1, from 1:1 to 3: 1, from 1:1 to 2: 1, from 2:1 to 5: 1, from 2: 1 to 4:1, from 2: 1 to 3:1, from 3:1 to 5: 1, from 3: 1 to 4:1, or from 4: 1 to 5:1. b. T cell Transduction

[0095] In certain embodiments, the methods of preparing engineered T cells disclosed herein comprise a step of introducing into a population of activated T cells a heterologous polynucleotide encoding a chimeric receptor, thereby generating a population of genetically modified T cells. This step may be referred to herein as “step (b).” The encoded chimeric receptor may be, for example, any of the chimeric receptors (e.g., CARs) described herein, including those described in section V hereinbelow. Any suitable method of introducing a heterologous polynucleotide may be used, including methods (either viral and non- viral) that result in the integration of the polynucleotide encoding the recombinant receptor into the genome of a T cell. Introduction of the heterologous polynucleotide encoding the chimeric receptor into the cell may be carried out using any of a number of known vectors, including viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, gammaretroviral vectors,etc.). Exemplary vectors and genetic engineering methods suitable for use in cell-preparation methods of the disclosure are described further in section V(f) hereinbelow.

[0096] In certain embodiments, the step of introducing the heterologous polynucleotide into the population of activated T cells is carried out in a basal culture medium. The basal culture medium may comprise, for example, TexMACS™ medium (Miltenyi Biotec) or OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher). In certain embodiments, the basal culture medium does not comprise serum. In certain embodiments, the basal culture medium is supplemented with one or more additional components for the maintenance, expansion, and / or activation of T cells (e.g., through supplementation with OpTmizer™ T-Cell Expansion Supplement (ThermoFisher)). In certain embodiments, the basal culture medium further comprises a serum replacement supplement, for example, an immune cell serum replacement, e.g., ThermoFisher, #A2596101, the CTS™ Immune Cell Serum Replacement, or the immune cell serum replacement described in Smith et al. (2015) CLIN. TRANSL. IMMUNOLOGY, 4(1): e31. In certain embodiments, the basal culture medium comprises a free form of an amino acid, such as L-glutamine. In certain embodiments, the basal culture medium comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In certain embodiments, the basal culture medium used is the same basal culture medium used in step (a).

[0097] In certain embodiments, the basal culture medium used in step (b) comprises one or more cytokines, e.g., recombinant cytokines. For example, in certain embodiments, the basal culture medium used in step (b) comprises wild-type or recombinant human IL-2, wild-type or recombinant human IL-7, and / or wild-type or recombinant human IL- 15. In certain embodiments, the basal culture medium used in step (b) comprises human IL-7 (e.g., wildtype or recombinant IL-7) and human IL-15 (e.g., wild-type or recombinant IL-15). In certain embodiments, the basal culture medium used in step (b) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL-15 (e.g., wild-type or recombinant IL-15) at a concentration of 1 to 100 ng / mL, e.g., 1 to 90 ng / mL, 1 to 80 ng / mL, 1 to 70 ng / mL, 1 to 60 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 35 ng / mL, 1 to 30 ng / mL, 1 to 25 ng / mL, 1 to 20 ng / mL, 1 to 15 ng / mL, 1 to 12.5 ng / mL, 1 to 10 ng / mL, 1 to 7.5 ng / mL, 1 to 5 ng / mL, 5 to 100 ng / mL, 5 to 90 ng / mL, 5 to 80 ng / mL, 5 to 70 ng / mL, 5 to 60 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 5 to 35 ng / mL, 5 to 30 ng / mL, 5 to 25 ng / mL, 5 to 20 ng / mL, 5 to 15 ng / mL, 5 to 12.5 ng / mL, 5 to 10 ng / mL, 5 to 7.5 ng / mL, 7.5 to 100 ng / mL, 7.5 to 90 ng / mL, 7.5 to 80 ng / mL, 7.5 to 70 ng / mL, 7.5 to 60 ng / mL, 7.5 to 50 ng / mL, 7.5 to 40 ng / mL, 7.5 to 35ng / mL, 7.5 to 30 ng / mL, 7.5 to 25 ng / mL, 7.5 to 20 ng / mL, 7.5 to 15 ng / mL, 7.5 to 12.5 ng / mL, 7.5 to 10 ng / mL, 10 to 100 ng / mL, 10 to 90 ng / mL, 10 to 80 ng / mL, 10 to 70 ng / mL, 10 to 60 ng / mL, 10 to 50 ng / mL, 10 to 40 ng / mL, 10 to 35 ng / mL, 10 to 30 ng / mL, 10 to 25 ng / mL, 10 to 20 ng / mL, 10 to 15 ng / mL, 10 to 12.5 ng / mL, 12.5 to 100 ng / mL, 12.5 to 90 ng / mL, 12.5 to 80 ng / mL, 12.5 to 70 ng / mL, 12.5 to 60 ng / mL, 12.5 to 50 ng / mL, 12.5 to 40 ng / mL, 12.5 to 35 ng / mL, 12.5 to 30 ng / mL, 12.5 to 25 ng / mL, 12.5 to 20 ng / mL, 12.5 to 15 ng / mL, 15 to 100 ng / mL, 15 to 90 ng / mL, 15 to 80 ng / mL, 15 to 70 ng / mL, 15 to 60 ng / mL, 15 to 50 ng / mL, 15 to 40 ng / mL, 15 to 35 ng / mL, 15 to 30 ng / mL, 15 to 25 ng / mL, 15 to 20 ng / mL, 20 to 100 ng / mL, 20 to 90 ng / mL, 20 to 80 ng / mL, 20 to 70 ng / mL, 20 to 60 ng / mL, 20 to 50 ng / mL, 20 to 40 ng / mL, 20 to 35 ng / mL, 20 to 30 ng / mL, 20 to 25 ng / mL, 25 to 100 ng / mL, 25 to 90 ng / mL, 25 to 80 ng / mL, 25 to 70 ng / mL, 25 to 60 ng / mL, 25 to 50 ng / mL, 25 to 40 ng / mL, 25 to 35 ng / mL, 25 to 30 ng / mL, 30 to 100 ng / mL, 30 to 90 ng / mL, 30 to 80 ng / mL, 30 to 70 ng / mL, 30 to 60 ng / mL, 30 to 50 ng / mL, 30 to 40 ng / mL, 30 to 35 ng / mL, 35 to 100 ng / mL, 35 to 90 ng / mL, 35 to 80 ng / mL, 35 to 70 ng / mL, 35 to 60 ng / mL, 35 to 50 ng / mL, 35 to 40 ng / mL, 40 to 100 ng / mL, 40 to 90 ng / mL, 40 to 80 ng / mL, 40 to 70 ng / mL, 40 to 60 ng / mL, 40 to 50 ng / mL, 50 to 100 ng / mL, 50 to 90 ng / mL, 50 to 80 ng / mL, 50 to 70 ng / mL, 50 to 60 ng / mL, 60 to 100 ng / mL, 60 to 90 ng / mL, 60 to 80 ng / mL, 60 to 70 ng / mL, 70 to 100 ng / mL, 70 to 90 ng / mL, 70 to 80 ng / mL, 80 to 100 ng / mL, 80 to 90 ng / mL, or 90 to 100 ng / mL. In certain embodiments, the basal culture medium used in step (b) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL-15 (e.g., wild-type or recombinant IL- 15) at a concentration of 10 to 15 ng / mL, e.g., at a concentration of 12.5 ng / mL. In certain embodiments, the basal culture medium used in step (b) comprises human IL-7 e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild-type or recombinant IL- 15) at a concentration of at least 1 ng / mL, at least 5 ng / mL, at least 10 ng / mL, at least 12.5 ng / mL, at least 15 ng / mL, at least 20 ng / mL, at least 25 ng / mL, at least 30 ng / mL, at least 35 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, or at least 100 ng / mL. In certain embodiments, the basal culture medium used in step (b) comprises human IL-7 (e.g., wildtype or recombinant IL-7) and / or human IL- 15 (e.g., wild-type or recombinant IL- 15) at a concentration of at least 10 ng / mL or at least 12.5 ng / mL. In certain embodiments, human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL-15 (e.g., wild-type or recombinant IL- 15) are used in step (b) at the same concentration as used in step (a).

[0098] In certain embodiments, the heterologous polynucleotide is introduced into the population of activated T cells in the presence of a transduction adjuvant. Exemplary transduction adjuvants include, but are not limited to, polycations, fibronectin or fibronectin- derived fragments or variants, and RetroNectin. In particular embodiments, the cells are engineered in the presence of a polycation selected from polybrene, DEAE-dextran, protamine sulfate, poly-L-lysine, or a cationic liposome.

[0099] In certain embodiments, the population of activated T cells is contacted with a vector (e.g., viral vector) comprising the heterologous polynucleotide, or incubated in the basal culture medium comprising the heterologous polynucleotide or vector, for 0.5 to 144 hours. For example, in certain embodiments, step (b) comprises contacting the population of activated T cells with the vector comprising the heterologous polynucleotide, or incubating the population of activated T cells in basal culture medium comprising the heterologous polynucleotide, for 1 to 144 hours, 1 to 120 hours, 1 to 96 hours, 1 to 72 hours, 1 to 60 hours, 1 to 54 hours, 1 to 48 hours, 1 to 40 hours, 1 to 36 hours, 1 to 32 hours, 1 to 28 hours, 1 to 24 hours, 1 to 20 hours, 1 to 18 hours, 1 to 16 hours, 1 to 14 hours, 1 to 12 hours, 1 to 10 hours,1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2 to 144 hours, 2 to 120 hours, 2 to 96 hours, 2 to 72 hours, 2 to 60 hours, 2 to 54 hours, 2 to 48 hours, 2 to 40 hours, 2 to 36 hours,2 to 32 hours, 2 to 28 hours, 2 to 24 hours, 2 to 20 hours, 2 to 18 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 144 hours, 4 to 120 hours, 4 to 96 hours, 4 to 72 hours, 4 to 60 hours, 4 to 54 hours, 4 to 48 hours, 4 to 40 hours, 4 to 36 hours, 4 to 32 hours, 4 to 28 hours, 4 to 24 hours, 4 to 20 hours, 4 to 18 hours, 4 to 16 hours, 4 to 14 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 144 hours, 6 to 120 hours, 6 to 96 hours, 6 to 72 hours, 6 to 60 hours, 6 to 54 hours, 6 to 48 hours, 6 to 40 hours, 6 to 36 hours, 6 to 32 hours, 6 to 28 hours, 6 to 24 hours, 6 to 20 hours, 6 to 18 hours, 6 to 16 hours, 6 to 14 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 144 hours, 8 to 120 hours, 8 to 96 hours, 8 to 72 hours, 8 to 60 hours, 8 to 54 hours, 8 to 48 hours, 8 to 40 hours, 8 to 36 hours, 8 to 32 hours, 8 to 28 hours, 8 to 24 hours, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 8 to 12 hours, 8 to 10 hours, 10 to 144 hours, 10 to 120 hours, 10 to 96 hours, 10 to 72 hours, 10 to 60 hours, 10 to 54 hours, 10 to 48 hours, 10 to 40 hours, 10 to 36 hours, 10 to 32 hours, 10 to 28 hours, 10 to 24 hours, 10 to 20 hours, 10 to 18 hours, 10 to 16 hours, 10 to 14 hours, 10 to 12 hours, 12 to 144 hours, 12 to 120 hours, 12 to 96 hours, 12 to 72 hours, 12 to 60 hours, 12 to 54 hours, 12 to 48 hours, 12 to 40 hours, 12 to 36 hours, 12 to 32 hours, 12 to 28 hours, 12 to 24 hours, 12 to 20 hours, 12 to 18 hours, 12 to16 hours, 12 to 14 hours, 14 to 144 hours, 14 to 120 hours, 14 to 96 hours, 14 to 72 hours, 14 to 60 hours, 14 to 54 hours, 14 to 48 hours, 14 to 40 hours, 14 to 36 hours, 14 to 32 hours, 14 to 28 hours, 14 to 24 hours, 14 to 20 hours, 14 to 18 hours, 14 to 16 hours, 16 to 144 hours, 16 to 120 hours, 16 to 96 hours, 16 to 72 hours, 16 to 60 hours, 16 to 54 hours, 16 to 48 hours, 16 to 40 hours, 16 to 36 hours, 16 to 32 hours, 16 to 28 hours, 16 to 24 hours, 16 to 20 hours, 16 to 18 hours, 18 to 144 hours, 18 to 120 hours, 18 to 96 hours, 18 to 72 hours, 18 to 60 hours, 18 to 54 hours, 18 to 48 hours, 18 to 40 hours, 18 to 36 hours, 18 to 32 hours, 18 to 28 hours, 18 to 24 hours, 18 to 20 hours, 20 to 144 hours, 20 to 120 hours, 20 to 96 hours, 20 to 72 hours, 20 to 60 hours, 20 to 54 hours, 20 to 48 hours, 20 to 40 hours, 20 to 36 hours, 20 to 32 hours, 20 to 28 hours, 20 to 24 hours, 24 to 144 hours, 24 to 120 hours, 24 to 96 hours, 24 to 72 hours, 24 to 60 hours, 24 to 54 hours, 24 to 48 hours, 24 to 40 hours, 24 to 36 hours, 24 to 32 hours, 24 to 28 hours, 28 to 144 hours, 28 to 120 hours, 28 to 96 hours, 28 to 72 hours, 28 to 60 hours, 28 to 54 hours, 28 to 48 hours, 28 to 40 hours, 28 to 36 hours, 28 to 32 hours, 32 to 144 hours, 32 to 120 hours, 32 to 96 hours, 32 to 72 hours, 32 to 60 hours, 32 to 54 hours, 32 to 48 hours, 32 to 40 hours, 32 to 36 hours, 36 to 144 hours, 36 to 120 hours, 36 to 96 hours, 36 to 72 hours, 36 to 60 hours, 36 to 54 hours, 36 to 48 hours, 36 to 40 hours, 40 to 144 hours, 40 to 120 hours, 40 to 96 hours, 40 to 72 hours, 40 to 60 hours, 40 to 54 hours, 40 to 48 hours, 48 to 144 hours, 48 to 120 hours, 48 to 96 hours, 48 to 72 hours, 48 to 60 hours, 48 to 54 hours, 54 to 144 hours, 54 to 120 hours, 54 to 96 hours, 54 to 72 hours, 54 to 60 hours, 60 to 144 hours, 60 to 120 hours, 60 to 96 hours, 60 to 72 hours, 72 to 144 hours, 72 to 120 hours, 72 to 96 hours, 96 to 144 hours, 96 to 120 hours, or 120 to 144 hours. In certain embodiments, step (b) comprises contacting the population of activated T cells with the vector comprising the heterologous polynucleotide, or incubating the population of activated T cells in basal culture medium comprising the heterologous polynucleotide, for at least 1 hour, e.g., at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 28 hours, at least 32 hours, at least 36 hours, at least 40 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours. In certain embodiments, step (b) comprises contacting the population of activated T cells with the vector comprising the heterologous polynucleotide, or incubating the population of activated T cells in basal culture medium comprising the heterologous polynucleotide, for 1 to 7 days, e.g. 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 7 days, 5 to 6days, or 6 to 7 days. In certain embodiments, step (b) comprises contacting the population of activated T cells with the vector comprising the heterologous polynucleotide, or incubating the population of activated T cells in basal culture medium comprising the heterologous polynucleotide, for at least 1 day, e.g., at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days.

[0100] In certain embodiments wherein the vector comprising the heterologous polynucleotide is a viral vector, the vector can be added to the basal culture media at, or used at, for example, a multiplicity of infection (MOI) of from 4 to 10. In certain embodiments, the viral vector is added or used at an MOI of from 4 and 10, e.g., from 4 to 9, from 4 to 8, from 4 to 7, from 4 to 6, from 4 to 5, from 5 to 10, from 5 to 9, from 5 to 8, from 5 to 7, from 5 to 6, from 6 to 10, from 6 to 9, from 6 to 8, from 6 to 7, from 7 to 10, from 7 to 9, from 7 to 8, from 8 to 10, from 8 to 9, or from 9 to 10. In certain embodiments, the viral vector is added or used at an MOI of, e.g., from 4 to 10 or from 7 to 9, e.g., 8. In certain embodiments, the viral vector is added or used at an MOI of at least 4, e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In certain embodiments, the viral vector is added or used at an MOI of at least 7 or 8. In certain embodiments, the viral vector is used at an MOI of no greater than 10, e.g., no greater than 9, no greater than 8, no greater than 7, no greater than 6, no greater than 5, or no greater than 4.

[0101] The step of introducing the heterologous polynucleotide into the population of activated T cells can optionally be carried out in any suitable cell processing system known in the art, including, but not limited to, the CliniMACS Prodigy™ system (Miltenyi Biotec). In certain embodiments, the step of introducing the heterologous polynucleotide (step (b)) is carried out in the same cell processing system as the step of T-cell activation (step (a)) is carried out in. In certain embodiments, the population of activated T cells was activated in a cell processing system (e.g. a CliniMACS Prodigy system (Miltenyi Biotec)), and the activated T cells are not removed from the system prior to initiating the step of introducing the heterologous polynucleotide. In certain embodiments, step (b) is initiated by adding the heterologous polynucleotide or vector (e.g., viral vector) comprising the heterologous polynucleotide directly to the basal culture medium used in step (a).

[0102] The step of introducing the heterologous polynucleotide into the population of activated T cells may optionally be carried out in any suitable bioreactor known in the art, including, but not limited to, a bioreactor comprising an oxygen-permeable membrane (e.g., a G-Rex™ bioreactor). In certain embodiments, the step of introducing the heterologouspolynucleotide (step (b)) is carried out in the same bioreactor as the step of T-cell activation (step (a)) is carried out in.

[0103] In certain embodiments, the population of activated T cells is contacted with the heterologous polynucleotide (or a vector comprising the same) within a certain period of time from when the sample (e.g. , whole blood sample) is isolated from the subject. For example, in certain embodiments, the population of primary T cells is contacted with the activating agent within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day of when the sample e.g., whole blood sample) is isolated from the subject. In certain embodiments, the population of activated T cells is contacted with the heterologous polynucleotide (or a vector comprising the same) within 72 hours, within 48 hours, or within 24 hours of when the sample (e.g., whole blood sample) is isolated from the subject.

[0104] In certain embodiments, the population of activated T cells is contacted with the heterologous polynucleotide (or a vector comprising the same) within a certain period of time from when the population of primary T cells is initially contacted with the activating agent and / or within some period of time from the initiation of step (a). For example, in certain embodiments, the population of activated T cells is contacted with the heterologous polynucleotide (or a vector comprising the same) within 7 days, within 6 days, within 5 days, within 4 days, within 72 hours, within 60 hours, within 54 hours, within 48 hours, within 40 hours, within 32 hours, within 28 hours, within 24 hours, within 22 hours, within 20 hours, within 18 hours, within 16 hours, within 15 hours, within 14 hours, within 13 hours, within 12 hours, within 10 hours, within 8 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, or within 1 hour of initially contacting the population of primary T cells with the activating agent and / or initiating step (a).

[0105] In certain embodiments, at the conclusion of step (b), at least some percentage T cells in the culture comprise the heterologous polynucleotide in their genome and / or express the chimeric receptor. Efficiency of introduction of the heterologous polynucleotide or expression of the chimeric receptor encoded by the heterologous polynucleotide can be measured using any suitable technique known in the art, including, but not limited to, by measuring the relative abundance of the heterologous polynucleotide in the population ofcells using quantitative PCR (qPCR), or by detecting the proportion of chimeric -receptor- expressing cells using, e.g., cell-sorting (e.g., fluorescence-activated cell sorting). In certain embodiments, at or after the completion of step (b), at least 10%, e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the T cells in the culture comprise the heterologous polynucleotide and / or express the chimeric receptor. In certain embodiments wherein the sample obtained from the subject is a whole blood sample, the efficiency of introduction of the heterologous polynucleotide (e.g. , the efficiency of transduction) is compared to that of an appropriate control population of activated T cells which was derived from an apheresis sample (e.g., a leukapheresis sample). The appropriate control population can, for example, be a population of activated T cell derived from an apheresis product, which (apart from being derived from a leukapheresis sample) is otherwise treated and activated in substantially the same way as the population of activated T cells derived from the whole sample. In certain embodiments, the whole blood sample and the apheresis product (e.g., leukapheresis product) are both obtained from the same subject. In certain embodiments, the efficiency of introduction of the heterologous polynucleotide into the whole-blood-sample-sourced population of T cells is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 140%, or at least 150% as efficient as the introduction of the heterologous polynucleotide into the population of the apheresis-product-sourced population of T cells. c. T-cell Expansion

[0106] In certain embodiments, the methods of preparing engineered T cells disclosed herein comprise a step of incubating a population of genetically modified T cells in a basal culture medium for at least 2 days, thereby generating a population of expanded T cells. This step may be referred to herein as “step (c).”

[0107] In certain embodiments, the basal culture medium may comprise, for example, TexMACS™ (Miltenyi Biotec) medium or OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher). In certain embodiments, the basal culture medium is supplemented with one or more additional components for the maintenance, expansion, and / or activation of T cells (e.g., through supplementation with OpTmizer™ T-Cell Expansion Supplement (ThermoFisher)). In certain embodiments, the basal culture medium further comprises aserum replacement supplement, for example, an immune cell serum replacement, e.g., ThermoFisher, #A2596101, the CTS™ Immune Cell Serum Replacement, or the immune cell serum replacement described in Smith et al. (2015) CLIN. TRANSL. IMMUNO OGY, 4(1): e31. In certain embodiments, the basal culture medium comprises a free form of an amino acid, such as L-glutamine. In certain embodiments, the basal culture medium comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In certain embodiments, the basal culture medium comprises serum. For example, the basal culture medium can comprise, e.g., human AB antibody serum (HABS). In certain embodiments, the basal culture medium used for T-cell expansion is the same basal culture medium used in step (a) and / or step (b). In certain embodiments, the basal culture medium used for T-cell expansion is the same basal culture medium used in step (a) and / or step (b), except that the basal culture medium is further supplemented with serum (e.g., HABS).

[0108] In certain embodiments, the basal culture medium used in step (c) comprises human serum (e.g., HABS), wherein the human serum is present in the culture medium at a concentration of at least 1% by volume. For example, in certain embodiments, human serum (e.g., HABS) is present in the basal culture medium at 1% to 10%, 1% to 7.5%, 1% to 5%, 1 % to 4%, 1% to 3%, 1% to 2%, 2% to 10%, 2% to 7.5%, 2% to 5%, 2% to 4%, 2% to 3%, 3% to 10%, 3% to 7.5%, 3% to 5%, 3% to 4%, 4% to 10%, 4% to 7.5%, 4% to 5%, 5% to 10%, 5% to 7.5%, or 7.5% to 10% by volume. In certain embodiments, human serum (e.g., HABS) is present in the basal culture medium at a concentration of at least 1%, at least 2%, at least 2.5%, at least 3%, at least 4%, at least 5%, at least 7.5%, or at least 10% by volume. In certain embodiments, human serum (e.g., HABS) is present in the culture medium at a concentration of at least 5% by volume.

[0109] In certain embodiments, the basal culture medium used in step (c) comprises one or more cytokines, e.g., recombinant cytokines. For example, in certain embodiments, the basal culture medium used in step (c) comprises wild-type or recombinant human IL-2, wild-type or recombinant human IL-7, and / or wild-type or recombinant human IL- 15. In certain embodiments, the basal culture medium used in step (c) comprises human IL-7 (e.g., wildtype or recombinant IL-7) and human IL-15 (e.g., wild-type or recombinant IL-15). In certain embodiments, the basal culture medium used in step (c) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild-type or recombinant IL- 15) at a concentration of 1 to 100 ng / mL, e.g., 1 to 90 ng / mL, 1 to 80 ng / mL, 1 to 70 ng / mL, 1 to 60ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 35 ng / mL, 1 to 30 ng / mL, 1 to 25 ng / mL, 1 to 20 ng / mL, 1 to 15 ng / mL, 1 to 12.5 ng / mL, 1 to 10 ng / mL, 1 to 7.5 ng / mL, 1 to 5 ng / mL, 5 to 100 ng / mL, 5 to 90 ng / mL, 5 to 80 ng / mL, 5 to 70 ng / mL, 5 to 60 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 5 to 35 ng / mL, 5 to 30 ng / mL, 5 to 25 ng / mL, 5 to 20 ng / mL, 5 to 15 ng / mL, 5 to 12.5 ng / mL, 5 to 10 ng / mL, 5 to 7.5 ng / mL, 7.5 to 100 ng / mL, 7.5 to 90 ng / mL, 7.5 to 80 ng / mL, 7.5 to 70 ng / mL, 7.5 to 60 ng / mL, 7.5 to 50 ng / mL, 7.5 to 40 ng / mL, 7.5 to 35 ng / mL, 7.5 to 30 ng / mL, 7.5 to 25 ng / mL, 7.5 to 20 ng / mL, 7.5 to 15 ng / mL, 7.5 to 12.5 ng / mL, 7.5 to 10 ng / mL, 10 to 100 ng / mL, 10 to 90 ng / mL, 10 to 80 ng / mL, 10 to 70 ng / mL,10 to 60 ng / mL, 10 to 50 ng / mL, 10 to 40 ng / mL, 10 to 35 ng / mL, 10 to 30 ng / mL, 10 to 25 ng / mL, 10 to 20 ng / mL, 10 to 15 ng / mL, 10 to 12.5 ng / mL, 12.5 to 100 ng / mL, 12.5 to 90 ng / mL, 12.5 to 80 ng / mL, 12.5 to 70 ng / mL, 12.5 to 60 ng / mL, 12.5 to 50 ng / mL, 12.5 to 40 ng / mL, 12.5 to 35 ng / mL, 12.5 to 30 ng / mL, 12.5 to 25 ng / mL, 12.5 to 20 ng / mL, 12.5 to 15 ng / mL, 15 to 100 ng / mL, 15 to 90 ng / mL, 15 to 80 ng / mL, 15 to 70 ng / mL, 15 to 60 ng / mL, 15 to 50 ng / mL, 15 to 40 ng / mL, 15 to 35 ng / mL, 15 to 30 ng / mL, 15 to 25 ng / mL, 15 to 20 ng / mL, 20 to 100 ng / mL, 20 to 90 ng / mL, 20 to 80 ng / mL, 20 to 70 ng / mL, 20 to 60 ng / mL, 20 to 50 ng / mL, 20 to 40 ng / mL, 20 to 35 ng / mL, 20 to 30 ng / mL, 20 to 25 ng / mL, 25 to 100 ng / mL, 25 to 90 ng / mL, 25 to 80 ng / mL, 25 to 70 ng / mL, 25 to 60 ng / mL, 25 to 50 ng / mL, 25 to 40 ng / mL, 25 to 35 ng / mL, 25 to 30 ng / mL, 30 to 100 ng / mL, 30 to 90 ng / mL, 30 to 80 ng / mL, 30 to 70 ng / mL, 30 to 60 ng / mL, 30 to 50 ng / mL, 30 to 40 ng / mL, 30 to 35 ng / mL, 35 to 100 ng / mL, 35 to 90 ng / mL, 35 to 80 ng / mL, 35 to 70 ng / mL, 35 to 60 ng / mL, 35 to 50 ng / mL, 35 to 40 ng / mL, 40 to 100 ng / mL, 40 to 90 ng / mL, 40 to 80 ng / mL, 40 to 70 ng / mL, 40 to 60 ng / mL, 40 to 50 ng / mL, 50 to 100 ng / mL, 50 to 90 ng / mL, 50 to 80 ng / mL, 50 to 70 ng / mL, 50 to 60 ng / mL, 60 to 100 ng / mL, 60 to 90 ng / mL, 60 to 80 ng / mL, 60 to 70 ng / mL, 70 to 100 ng / mL, 70 to 90 ng / mL, 70 to 80 ng / mL, 80 to 100 ng / mL, 80 to 90 ng / mL, or 90 to 100 ng / mL. In certain embodiments, the basal culture medium used in step (c) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild-type or recombinant IL-15) at a concentration of 10 to 15 ng / mL, e.g., at a concentration of 12.5 ng / mL. In certain embodiments, the basal culture medium used in step (c) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL-15 (e.g., wild-type or recombinant IL- 15) at a concentration of at least 1 ng / mL, at least 5 ng / mL, at least 10 ng / mL, at least 12.5 ng / mL, at least 15 ng / mL, at least 20 ng / mL, at least 25 ng / mL, at least 30 ng / mL, at least 35 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, or at least 100 ng / mL. In certain embodiments, the basal culture medium used in step (c) comprises human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL- 15 (e.g., wild- type or recombinant IL- 15) at a concentration of at least 10 ng / mL or at least 12.5 ng / mL. In certain embodiments, human IL-7 (e.g., wild-type or recombinant IL-7) and / or human IL-15 (e.g., wild-type or recombinant IL- 15) are used in step (c) at the same concentration as used in step (a) and / or step (b).

[0110] The population of genetically modified T cells can be expanded in a volume of the basal culture medium ranging from, for example, 50 mL to 2 L, e.g., 50 mL to 1.75 L, 50 mL to 1.5 L, 50 mL to 1.25 L, 50 mL to 1 L, 50 mL to 750 mL, 50 mL to 500 mL, 50 mL to 250 mL, 50 mL to 200 mL, 50 mL to 150 mL, 50 mL to 100 mL, 50 mL to 70 mL, 70 mL to 2 L, 70 mL to 1.75 L, 70 mL to 1.5 L, 70 mL to 1.25 L, 70 mL to 1 L, 70 mL to 750 mL, 70 mL to 500 mL, 70 mL to 250 mL, 70 mL to 200 mL, 70 mL to 150 mL, 70 mL to 100 mL, 100 mL to 2 L, 100 mL to 1.75 L, 100 mL to 1.5 L, 100 mL to 1.25 L, 100 mL to 1 L, 100 mL to 750 mL, 100 mL to 500 mL, 100 mL to 250 mL, 100 mL to 200 mL, 100 mL to 150 mL, 150 mL to 2 L, 150 mL to 1.75 L, 150 mL to 1.5 L, 150 mL to 1.25 L, 150 mL to 1 L, 150 mL to 750 mL, 150 mL to 500 mL, 150 mL to 250 mL, 150 mL to 200 mL, 200 mL to 2 L, 200 mL to1.75 L, 200 mL to 1.5 L, 200 mL to 1.25 L, 200 mL to 1 L, 200 mL to 750 mL, 200 mL to 500 mL, 200 mL to 250 mL, 250 mL to 2 L, 250 mL to 1.75 L, 250 mL to 1.5 L, 250 mL to 1.25 L, 250 mL to 1 L, 250 mL to 750 mL, 250 mL to 500 mL, 500 mL to 2 L, 500 mL to1.75 L, 500 mL to 1.5 L, 500 mL to 1.25 L, 500 mL to 1 L, 500 mL to 750 mL, 750 mL to 2 L, 750 mL to 1.75 L, 750 mL to 1.5 L, 750 mL to 1.25 L, 750 mL to 1 L, 1 L to 2 L, 1 L to1.75 L, 1 L to 1.5 L, 1 L to 1.25 L, 1.25 L to 2 L, 1.25 L to 1.75 L, 1.25 L to 1.5 L, 1.5 L to 2 L, 1.5 L to 1.75 L, or 1.75 L to 2 L. In certain embodiments, the population of genetically modified T cells is expanded in a volume of the basal culture medium ranging from 100 mL to IL. In certain embodiments, the population of genetically modified T cells is expanded in at least a volume of 50 mL, 70 mL, 100 mL, 150 mL, 200 mL, 250 mL, 500 mL, 750 mL, 1 L, 1.25 L, 1.5 L, 1.75 L, or 2 L of the basal culture medium. In certain embodiments wherein T-cell expansion is carried out in a bioreactor (e.g., a G-Rex™ bioreactor), the genetically modified T cells are expanded in a volume of basal culture medium that is suitable for use in the bioreactor.

[0111] In certain embodiments, the expansion step comprises incubating the population of genetically modified T cells in the basal culture medium for at least 2 days. For example, in certain embodiments, the expansion step comprises incubating the population of genetically modified T cells in the basal culture medium for 2 to 14 days, 2 to 12 days, 2 to 10 days, 2 to9 days, 2 to 8 days, 2 to 7.5 days, 2 to 7 days, 2 to 6.5 days, 2 to 6 days, 2 to 5.5 days, 2 to 5 days, 2 to 4.5 days, 2 to 4 days, 2 to 3.5 days, 2 to 3 days, 2 to 2.5 days, 2.5 to 14 days, 2.5 to 12 days, 2.5 to 10 days, 2.5 to 9 days, 2.5 to 8 days, 2.5 to 7.5 days, 2.5 to 7 days, 2.5 to 6.5 days, 2.5 to 6 days, 2.5 to 5.5 days, 2.5 to 5 days, 2.5 to 4.5 days, 2.5 to 4 days, 2.5 to 3.5 days, 2.5 to 3 days, 3 to 14 days, 3 to 12 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7.5 days, 3 to 7 days, 3 to 6.5 days, 3 to 6 days, 3 to 5.5 days, 3 to 5 days, 3 to 4.5 days, 3 to 4 days, 3 to 3.5 days, 3.5 to 14 days, 3.5 to 12 days, 3.5 to 10 days, 3.5 to 9 days, 3.5 to 8 days,3.5 to 7.5 days, 3.5 to 7 days, 3.5 to 6.5 days, 3.5 to 6 days, 3.5 to 5.5 days, 3.5 to 5 days, 3.5 to 4.5 days, 3.5 to 4 days, 4 to 14 days, 4 to 12 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7.5 days, 4 to 7 days, 4 to 6.5 days, 4 to 6 days, 4 to 5.5 days, 4 to 5 days, 4 to 4.5 days, 4.5 to 14 days, 4.5 to 12 days, 4.5 to 10 days, 4.5 to 9 days, 4.5 to 8 days, 4.5 to 7.5 days, 4.5 to 7 days, 4.5 to 6.5 days, 4.5 to 6 days, 4.5 to 5.5 days, 4.5 to 5 days, 5 to 14 days, 5 to 12 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7.5 days, 5 to 7 days, 5 to 6.5 days, 5 to 6 days, 5 to5.5 days, 5.5 to 14 days, 5.5 to 12 days, 5.5 to 10 days, 5.5 to 9 days, 5.5 to 8 days, 5.5 to 7.5 days, 5.5 to 7 days, 5.5 to 6.5 days, 5.5 to 6 days, 6 to 14 days, 6 to 12 days, 6 to 10 days, 6 to9 days, 6 to 8 days, 6 to 7.5 days, 6 to 7 days, 6 to 6.5 days, 6.5 to 14 days, 6.5 to 12 days, 6.5 to 10 days, 6.5 to 9 days, 6.5 to 8 days, 6.5 to 7.5 days, 6.5 to 7 days, 7 to 14 days, 7 to 12 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, 7 to 7.5 days, 7.5 to 14 days, 7.5 to 12 days, 7.5 to10 days, 7.5 to 9 days, 7.5 to 8 days, 8 to 14 days, 8 to 12 days, 8 to 10 days, 8 to 9 days, 9 to 14 days, 9 to 12 days, 9 to 10 days, 10 to 14 days, 10 to 12 days, or 12 to 14 days. In certain embodiments, the expansion step comprises incubating the population of genetically modified T cells in the basal culture medium for 2 to 10 days, 3 to 9 days, 4 to 8 days, or 5 to 7 days. In certain embodiments, the expansion step comprises incubating the population of genetically modified T cells in the basal culture medium for at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, or at least 14 days. For example, in certain embodiments, the expansion step comprises incubating the population of genetically modified T cells in the basal culture medium for at least 2 days, at least 3 days, or at least 6 days. In certain embodiments, the expansion step comprises incubating the population of genetically modified T cells in the basal culture medium for no more than 2 days, no more than 2.5 days, no more than 3 days, no more than 3.5 days, no more than 4 days, no more than 4.5 days, no more than 5 days, no more than 5.5 days, no more than 6 days, no morethan 6.5 days, no more than 7 days, no more than 7.5 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 12 days, or no more than 14 days.

[0112] In certain embodiments, the step of expanding the population of genetically modified T cells is initiated within a certain of time from when the sample (e.g. whole blood sample) is isolated from the subject. For example, in certain embodiments, the step of expanding the population of genetically modified T cells is initiated within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days within 5 days, within 4 days, within 3 days, or within 2 days of when the sample e.g., whole blood sample) is isolated from the subject.

[0113] In certain embodiments, the step of expanding the population of genetically modified T cells is initiated within a certain period of time from when the population of primary T cells is initially contacted with the activating agent and / or within a certain period of time from the initiation of step (a). For example, in certain embodiments, the step of expanding the population of genetically modified T cells is initiated within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, or within 2 days of initially contacting the population of primary T cells with the activating agent and / or initiating step (a). In certain embodiments, the step of expanding the population of genetically modified T cells is initiated within 4 days or within 3 days of initially contacting the population of primary T cells with the activating agent and / or initiating step (a).

[0114] In certain embodiments, the step of expanding the population of genetically modified T cells is initiated within a certain period of time from when the population of activated T cells is initially contacted with the heterologous polynucleotide or a vector comprising the same, and / or within a certain period of time from the initiation of step (b). For example, in certain embodiments, the step of expanding the population of genetically modified T cells is initiated within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day of initially contacting the population of activated T cells with the heterologous polynucleotide or vector comprising the same and / or initiating step (b). In certain embodiments, the step of expanding the population of genetically modified T cells is initiated within 3 days or within 2days of initially contacting the population of activated T cells with the heterologous polynucleotide or vector comprising the same and / or initiating step (b).

[0115] The step of expanding the population of genetically modified T cells can optionally be carried out in any suitable bioreactor known in the art, including, but not limited to, a bioreactor comprising an oxygen-permeable membrane (e.g., a G-Rex ™ bioreactor). In certain embodiments, one or both of steps (a) (T-cell activation) and (b) (introduction of the heterologous polynucleotide) is carried out in a cell processing system (e.g. a CliniMACS Prodigy™ system (Miltenyi Biotec)), and the population of genetically modified T cells is subsequently transferred to the bioreactor at the initiation of the T-cell expansion step. In certain embodiments, the population of genetically modified T cells is cultured in the basal culture medium in the bioreactor for the duration of the T-cell expansion step. In certain embodiments, T-cell expansion is carried out in a static culture bag.

[0116] In certain embodiments, the population of genetically modified T cells expands by at least 10-fold during the step of T-cell expansion (step (c)), e.g., after 6 days of incubating the cells in the basal culture medium. For example, in certain embodiments, the population of genetically modified T cells expands by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70- fold, or at least 75-fold during the step of T-cell expansion (“step (c)”), e.g., after 6 days of incubating the cells in the basal culture medium. In certain embodiments wherein the sample obtained from the subject is a whole blood sample, the fold-expansion of the population of T cells is compared to that of an appropriate control population of genetically modified T cells which was derived from an apheresis sample (e.g., a leukapheresis sample). The appropriate control population can, for example, be a population of genetically modified T cells derived from an apheresis product, which (apart from being derived from an apheresis product) is otherwise treated, activated, and expanded in substantially the same way as the population of genetically modified T cells derived from the whole blood sample. In certain embodiments, the whole blood sample and the apheresis product (e.g., leukapheresis product) are both obtained from the same subject. In certain embodiments, the fold-expansion of the wholeblood-sample-sourced population of T cells is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 140%, or at least 150% of the fold-expansion of the population of the apheresis-product-sourced sample of cells in the sameamount of time (e.g., as measured after each population of cells has been expanded for at least 6 days).

[0117] In certain embodiments, the step of expanding the population of genetically modified T cells (“step (c)”) yields at least 1 x 107genetically modified T cells e.g., after 6 days of incubating the population of genetically modified T cells in the basal culture medium. For example, in certain embodiments, the step of expanding the population of genetically modified T cells yields from 1 x 107to 1 x 109, from 1 x 107to 8 x 108, from 1 x 107to 6 x 10s, from 1 x 107to 5 x 108, from 1 x 107to 4 x 10s, from 1 x 107to 3 x 10s, from 1 x 107to 2 x 108, from 1 x 107to 1 x 10s, from 1 x 107to 5 x 107, from 5 x 107to 1 x 109, from 5 x 107to 8 x 108, from 5 x 107to 6 x 10s, from 5 x 107to 5 x 108, from 5 x 107to 4 x 108, from 5 x 107to 3 x 10s, from 5 x 107to 2 x 10s, from 5 x 107to 1 x 108, from 1 x 108to 1 x 109, from 1 x108to 8 x 10s, from 1 x 108to 6 x 108, from 1 x 108to 5 x 10s, from 1 x 108to 4 x 10s, from 1 x 108to 3 x 10s, from 1 x 10sto 2 x 108, from 2 x 108to 1 x 109, from 2 x 108to 8 x 10s, from2 x 108to 6 x 108, from 2 x 10sto 5 x 108, from 2 x 108to 4 x 108, from 2 x 108to 3 x 108, from 3 x 108to 1 x 109, from 3 x 108to 8 x 108, from 3 x 108to 6 x 108, from 3 x 108to 5 x 108, from 3 x 10sto 4 x 10s, from 4 x 108to 1 x 109, from 4 x 108to 8 x 10s, from 4 x 108to 6 x 10s, from 4 x 108to 5 x 10s, from 5 x 108to 1 x 109, from 5 x 108to 8 x 10s, from 5 x 108to 6 x 108, from 6 x 108to 1 x 109, from 6 x 10sto 8 x 108, or from 8 x 10sto 1 x 109genetically modified T cells (e.g., after 6 days of incubating the genetically modified T cells in the basal culture medium). In certain embodiments, the step of expanding the population of genetically modified T cells yields from 1 x 108to 3 x 108genetically modified T cells, or from 1 x 108to 2 x 108genetically modified T cells e.g., after 6 days of incubating the genetically modified T cells in the basal culture medium).

[0118] In certain embodiments wherein the sample obtained from the subject is a whole blood sample, the yield of the population of genetically T cells is compared to that of an appropriate control population of genetically modified T cells which was derived from an apheresis sample (e.g., a leukapheresis sample). The appropriate control population can, for example, be a population of genetically modified T cells derived from an apheresis product, which (apart from being derived from an apheresis product) is otherwise treated, activated, and expanded in substantially the same way as the population of genetically modified T cells derived from the whole blood sample. In certain embodiments, the whole blood sample and the apheresis product (e.g., leukapheresis product) are both obtained from the same subject. In certain embodiments, the yield of the whole-blood-sample-sourced population of T cells isat least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 1 10%, at least 115%, at least 120%, at least 125%, at least 130%, at least 140%, or at least 150% of the yield of the population of the apheresis- product- sourced sample of cells (e.g., after 6 days of incubating each population of genetically modified T cells in the basal culture medium). d. T-cell harvesting

[0119] In certain embodiments, the methods of preparing engineered T cells disclosed herein comprise a step of harvesting or collecting an expanded population of genetically modified T cells, thereby generating a composition comprising a population of engineered T cells. This step may be referred to herein as “step (d).”

[0120] In certain embodiments, the step of harvesting the population of expanded T cells is carried out within a certain period of time from when the sample (e.g., whole blood sample) is isolated from the subject. For example, in certain embodiments, the step of harvesting the population of expanded T cells is carried out within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, within 2 weeks, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days within 5 days, within 4 days, or within 3 days of when the sample e.g., whole blood sample) is isolated from the subject.

[0121] In certain embodiments, the step of harvesting the population of expanded T cells is carried out within a certain period of time from when the population of primary T cells is initially contacted with the activating agent and / or within a certain period of time from the initiation of step (a). For example, in certain embodiments, the step of harvesting the population of expanded T cells is carried out within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, or within 3 days of initially contacting the population of primary T cells with the activating agent and / or initiating step (a). In certain embodiments, the step of harvesting the population of expanded T cells is carried out within 10 days or within 9 days of initially contacting the population of primary T cells with the activating agent and / or initiating step (a).

[0122] In certain embodiments, the step of harvesting the population of expanded T cells is carried out within a certain period of time from when the population of activated T cells is initially contacted with the heterologous polynucleotide or a vector comprising the same, and / or within a certain period of time from the initiation of step (b). For example, in certain embodiments, the step of harvesting the population of expanded T cells is carried out within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, or within 2 days of initially contacting the population of activated T cells with the heterologous polynucleotide or vector comprising the same and / or initiating step (b). In certain embodiments, the step of harvesting the population of expanded T cells is carried out within 5 days, within 8 days, or within 9 days of initially contacting the population of activated T cells with the heterologous polynucleotide or vector comprising the same and / or initiating step (b).

[0123] In certain embodiments, the step of harvesting the population of expanded T cells is carried out within a certain period of time from when expansion of the population of genetically modified T cells is initiated, and / or a certain period of time from the initiation of step (c). For example, in certain embodiments, the step of harvesting the population of expanded T cells is carried out within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, or within 2 days from when expansion of the population of genetically modified T cells is initiated, and / or from the initiation of step (c). In certain embodiments, the step of harvesting the population of expanded T cells is carried out within 3 days, within 6 days, or within 7 days of when expansion of the population of genetically modified T cells is initiated, and / or the initiation of step (c).

[0124] In certain embodiments, harvesting or collecting the expanded population of T cells comprises a step of filtering the cell composition during or after the harvesting or collecting, e.g., using a filter, for example, to remove large particulates. In certain embodiments, the filtering step is performed while the cells are being harvested or collected. For example, a filter may be in-line with between the cells being incubated after expansion and a harvesting / collection device such as a cell processing system (e.g., the Sepax® or Sepax 2® cell processing systems). In certain embodiments, the cells are harvested or collected and then filtered before the filtered composition is optionally washed. In certain embodiments, the cells are harvested or collected and subsequently washed. In certain embodiments, the washed cell composition is then filtered.

[0125] In certain embodiments, the composition of engineered T cells is frozen (e.g., cryopreserved) after being harvested and prior to proceeding with formulation and / or administration to a subject. The cryopreservation solution or buffer can comprise, for example, a DMSO solution, optionally comprising human serum albumin (HSA), or another suitable cell freezing media. In certain embodiments, the cryopreserved composition is stored for a period of time greater than or equal to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In certain embodiments, the composition is placed into long-term storage. In certain embodiments, the composition is stored for a period of time greater than or equal to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more. In certain embodiments, the cryopreserved composition is stored for a period of time less than or equal to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0126] An engineered T-cell composition that has been frozen (e.g., cryopreserved) should be thawed prior to administration to a subject in need thereof. Thawing can occur, e.g., at a temperature of 34-40 °C, e.g., 34-40 °C, 34-39 °C, 34-38 °C, 34-37 °C, 34-36 °C, 34-35 °C, 35-40 °C, 35-39 °C, 35-38 °C, 35-37 °C, 35-36 °C, 36-40 °C, 36-39 °C, 36-38 °C, 36-37 °C, 37-40 °C, 37-39 °C, 37-38 °C, 38-40 °C, 38-39 °C, or 39-40 °C. In certain embodiments, the frozen sample of engineered T cells is thawed at 37 °C. Thawing may occur, e.g., in a heated system or water bath suitable for the purpose, e.g., a Plasma-therm™.

[0127] In certain embodiments, the engineered T cell composition is not frozen or cryopreserved prior to administration to a subject. In certain embodiments, the engineered T cell composition is never frozen or cryopreserved. In certain embodiments wherein the composition of engineered T cells is not frozen, the engineered T cells may be stored or transported at a temperature from 0 °C to 22 °C, e.g., from 0 °C to 20 °C, from 0 °C to 18 °C, from 0 °C to 16 °C, from 0 °C to 14 °C, from 0 °C to 12 °C, from 0 °C to 10 °C, from 0 °C to 8 °C, from 0 °C to 6 °C, from 0 °C to 4 °C, from 0 °C to 2 °C, from 2 °C to 22 °C, from 2 °Cto 20 °C, from 2 °C to 18 °C, from 2 °C to 16 °C, from 2 °C to 14 °C, from 2 °C to 12 °C, from 2 °C to 10 °C, from 2 °C to 8 °C, from 2 °C to 6 °C, from 2 °C to 4 °C, from 4 °C to 22 °C, from 4 °C to 20 °C, from 4 °C to 18 °C, from 4 °C to 16 °C, from 4 °C to 14 °C, from 4 °C to 12 °C, from 4 °C to 10 °C, from 4 °C to 8 °C, from 4 °C to 6 °C, from 6 °C to 22 °C, from 6 °C to 20 °C, from 6 °C to 18 °C, from 6 °C to 16 °C, from 6 °C to 14 °C, from 6 °C to 12 °C, from 6 °C to 10 °C, from 6 °C to 8 °C, from 8 °C to 22 °C, from 8 °C to 20 °C, from 8 °C to 18 °C, from 8 °C to 16 °C, from 8 °C to 14 °C, from 8 °C to 12 °C, from 8 °C to 10 °C, from 10 °C to 22 °C, from 10 °C to 20 °C, from 10 °C to 18 °C, from 10 °C to 16 °C, from 10 °C to 14 °C, from 10 °C to 12 °C, from 12 °C to 22 °C, from 12 °C to 20 °C, from 12 °C to 18 °C, from 12 °C to 16 °C, from 12 °C to 14 °C, from 14 °C to 22 °C, from 14 °C to 20 °C, from 14 °C to 18 °C, from 14 °C to 16 °C, from 16 °C to 22 °C, from 16 °C to 20 °C, from 16 °C to 18 °C, from 18 °C to 22 °C, from 18 °C to 20 °C, or from 20 °C to 22 °C. In certain embodiments wherein the engineered T cell composition is not frozen, the composition may be stored or transported at a temperature no greater than 22 °C, e.g., no greater than 20 °C, 18 °C, 16 °C, 14 °C, 12 °C, 10 °C, 8 °C, 6 °C, 4 °C, 2 °C, or 0 °C.

[0128] In certain embodiments, the T cell composition is administered to the subject (e.g., the same subject from whom the sample, (e.g., whole blood sample) was originally taken) within 3 months of harvesting the engineered T cells, e.g., within 3 months, 2 months, 1 month, 30 days, 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of harvesting the engineered T cells. In certain embodiments, the T cell composition is administered to the subject (e.g., the same subject from whom the sample (e.g., whole blood sample) was originally taken) within 6 months of isolating the whole blood sample from the subject, e.g., within 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 30 days, 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 1 1 days, 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days of isolating the sample (e.g., whole blood sample) from the subject. e. Additional steps

[0129] In certain embodiments, the methods of the disclosure comprise a step of (I) obtaining a population of PBMCs obtained from a whole blood sample isolated from a subject, and a step of (II) enriching for CD4+, CD8+, and / or CD3+ T cells in the populationof PBMCs. The enriched population of cells may then be used as the population of primary T cells to be activated in step (a).

[0130] In certain embodiments, the step of obtaining a population of PBMCs comprises thawing a frozen sample of PBMCs. Accordingly, in certain embodiments, the PBMCs have been previously frozen, e.g. , cryopreserved, and optionally stored for some period of time. A frozen sample of PBMCs can be thawed, e.g., at a temperature of 34-40 °C, e.g., 34-40 °C,34-39 °C, 34-38 °C, 34-37 °C, 34-36 °C, 34-35 °C, 35-40 °C, 35-39 °C, 35-38 °C, 35-37 °C,35-36 °C, 36-40 °C, 36-39 °C, 36-38 °C, 36-37 °C, 37-40 °C, 37-39 °C, 37-38 °C, 38-40 °C, 38-39 °C, or 39-40 °C. In certain embodiments, the frozen sample of PBMCs is thawed at 37 °C. Thawing may occur, e.g., in a heated system or water bath suitable for the purpose, e.g., a Plasma-therm™. A thawed population of PBMCs may additionally be washed prior to proceeding with enrichment and / or activation. In certain embodiments, the PBMCs are not frozen or cryopreserved prior to enrichment or activation. For example, in certain embodiments, the PMBCs have never been frozen or cryopreserved.

[0131] In certain embodiments, the PBMCs are obtained and subjected to enrichment within 1 month of isolating the whole blood sample from the subject, e.g., within 30 days, 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of isolating the whole blood sample from the subject. In certain embodiments wherein the PBMCs are not frozen, the PBMCs may be stored or transported at a temperature from 0 °C to 22 °C, e.g., from 0 °C to 20 °C, from 0 °C to 18 °C, from 0 °C to 16 °C, from 0 °C to 14 °C, from 0 °C to 12 °C, from 0 °C to 10 °C, from 0 °C to 8 °C, from 0 °C to 6 °C, from 0 °C to 4 °C, from 0 °C to 2 °C, from 2 °C to 22 °C, from 2 °C to 20 °C, from 2 °C to 18 °C, from 2 °C to 16 °C, from 2 °C to 14 °C, from 2 °C to 12 °C, from 2 °C to 10 °C, from 2 °C to 8 °C, from 2 °C to 6 °C, from 2 °C to 4 °C, from 4 °C to 22 °C, from 4 °C to 20 °C, from 4 °C to 18 °C, from 4 °C to 16 °C, from 4 °C to 14 °C, from 4 °C to 12 °C, from 4 °C to 10 °C, from 4 °C to 8 °C, from 4 °C to 6 °C, from 6 °C to 22 °C, from 6 °C to 20 °C, from 6 °C to 18 °C, from 6 °C to 16 °C, from 6 °C to 14 °C, from 6 °C to 12 °C, from 6 °C to 10 °C, from 6 °C to 8 °C, from 8 °C to 22 °C, from 8 °C to 20 °C, from 8 °C to 18 °C, from 8 °C to 16 °C, from 8 °C to 14 °C, from 8 °C to 12 °C, from 8 °C to 10 °C, from 10 °C to 22 °C, from 10 °C to 20 °C, from 10 °C to 18 °C, from 10 °C to 16 °C, from 10 °C to 14 °C, from 10 °C to 12 °C, from 12 °C to 22 °C, from 12 °C to 20 °C, from 12 °C to 18 °C, from 12 °C to 16 °C, from 12 °C to 14 °C, from 14 °C to 22 °C,from 14 °C to 20 °C, from 14 °C to 18 °C, from 14 °C to 16 °C, from 16 °C to 22 °C, from 16 °C to 20 °C, from 16 °C to 18 °C, from 18 °C to 22 °C, from 18 °C to 20 °C, or from 20 °C to 22 °C. In certain embodiments wherein the PBMCs are not frozen, the PBMCs may be stored or transported at a temperature no greater than 22 °C, e.g., no greater than 20 °C, 18 °C, 16 °C, 14 °C, 12 °C, 10 °C, 8 °C, 6 °C, 4 °C, 2 °C, or 0 °C.

[0132] In certain embodiments, PBMCs are obtained from the whole blood sample using density-based cell- separation methods. For example, in certain embodiments, PBMCs are obtained from the whole blood sample using density gradient centrifugation, e.g., centrifugation through a Ficoll gradient or a Percoll gradient. A different density gradient medium with the same density or greater density as Ficoll or Percoll may also be used, e.g., a density gradient medium having a density greater than 1.077 g / mL, e.g., greater than 1.1 g / mL, greater than 1.15 g / mL, greater than 1.2 g / mL, greater than 1.25 g / mL, greater than 1.3 g / mL, or greater than 1.31 g / mL. In certain embodiments, the density gradient medium comprises iodixanol, e.g., about 60% iodixanol in water. In certain embodiments, the density gradient medium OptiPrep™ (Sigma) may be used in density gradient centrifugation.Density gradient centrifugation can be performed in a cell separation device, e.g., a Sepax® or Sepax 2® cell processing system (Biosafe).

[0133] In certain embodiments, PBMCs are obtained from the whole blood sample by adding a red-blood-cell lytic agent to the whole blood sample. In certain embodiments, the lytic agent is water or an appropriate lysis buffer, e.g., ammonium-chloride-potassium (ACK) lysis buffer (ThermoFisher).

[0134] In certain embodiments, the enrichment step comprises positively selecting for certain cell markers, e.g., T cell markers such as CD3, CD4, and / or CD8. In certain embodiments, the enrichment step comprises selecting for CD4+ cells and CD8+ cells. This can be accomplished, e.g., via positive T cell selection with a combination of anti-CD4 and anti-CD8 magnetic beads (e.g., Miltenyi beads or Dynabeads®), optionally using automated column technology known in the art (such as CliniMACS® Plus or CliniMACS® Prodigy® systems). Alternatively, column-free technology with releasable CD3 beads (GE Healthcare) can be used. In addition, bead-free technologies such as ThermoGenesis X- series devices can be utilized as well. The enrichment step may also comprise, additionally or alternatively, negatively selecting for certain cell markers, e.g., markers of unwanted cells. Enrichment of a T cell population by negative selection can be accomplished, for example, with a combination of antibodies directed to surface markers unique to the negatively selected cells.This can be done, e.g., with cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. In certain embodiments, the positive or negative selection methods are performed under flow conditions, for example, by using a flow-through device or a cell processing system.

[0135] In certain embodiments, methods of the disclosure comprise a step of (I) enriching for CD4+, CD8+, and / or CD3+ T cells in the whole blood sample. The enriched population of cells obtained from the whole blood sample may then be used as the population of primary T cells to be activated in step (a).

[0136] In certain embodiments, the step of enriching for T cells in the whole blood sample comprises positively selecting for certain cell markers, e.g., T cell markers such as CD3, CD4, and / or CD8. In certain embodiments, the enrichment step comprises selecting for CD4+ cells and CD8+ cells. This can be accomplished, e.g., via positive T cell selection with a combination of anti-CD4 and anti-CD8 magnetic beads (e.g., Miltenyi beads or Dynabeads®), optionally using automated column technology known in the art (such as CliniMACS® Plus or CliniMACS® Prodigy® systems). Alternatively, column-free technology with releasable CD3 beads (GE Healthcare) can be used. In addition, bead-free technologies such as ThermoGenesis X- series devices can be utilized as well. The enrichment step may also comprise, additionally or alternatively, negatively selecting for certain cell markers, e.g., markers of unwanted cells. Enrichment of a T cell population by negative selection can be accomplished, for example, with a combination of antibodies directed to surface markers unique to the negatively selected cells. This can be done, e.g., with cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. In certain embodiments, the positive or negative selection methods are performed under flow conditions, for example, by using a flow-through device or a cell processing system.

[0137] In certain embodiments, the whole blood sample used in any of the foregoing methods is frozen (e.g., cryopreserved) after being obtained from the subject, and prior to proceeding with cell separation or T-cell enrichment steps. In certain embodiments, the population of PBMCs obtained through any of the foregoing methods is frozen (e.g., cryopreserved) prior to proceeding to the step of T-cell enrichment or activation. In certain embodiments, the enriched population of primary T cells to be used for T-cell activation isfrozen (e.g., cryopreserved) prior to proceeding with T-cell enrichment (step (a)). In any of these embodiments, the cryopreservation solution or buffer can comprise, for example, a DMSO solution, optionally comprising human serum albumin (HSA), or another suitable cell freezing media. In certain embodiments, the cryopreserved sample (e.g., whole blood sample, PBMC sample, or enriched T-cell sample) is stored for a period of time greater than or equal to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month. In certain embodiments, the sample is placed into long-term storage. In some aspects, the sample is stored for a period of time greater than or equal to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more. In certain embodiments, the cryopreserved sample is stored for a period of time less than or equal to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.III. Therapeutic Compositions

[0138] Engineered T cells produced using a method of the disclosure may be used to treat various diseases and disorders in a subject. In certain embodiments, the engineered T cells comprise CD8+ T cells. In certain embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the T cell composition are CD8+ T cells. In certain embodiments, the T 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. In certain embodiments, the ratio of CD4:CD8 T cells administered to the subject is from 1:5 to 5: 1, e.g. , from 1 :5 to 5 : 1 , from 1 :5 to 4: 1 , from 1 :5 to 3 : 1 , from 1 :5 to 2: 1 , from 1 :5 to 1:1, from 1 :5 to 1:2, from 1:5 to 1:3, from 1:5 to 1:4, from 1:4 to 5: 1, from 1:4 to 4:1 , from 1:4 to 3:1, from 1:4 to 2: 1, from 1:4 to 1 : 1, from 1:4 to 1:2, from 1:4 to 1:3, from 1:3 to 5:1, from 1:3 to 4:1, from 1:3 to 3: 1, from 1 :3 to 2:1, from 1:3 to 1:1, from 1 :3 to 1:2, from 1 :2 to 5:1, from 1:2 to 4: 1, from 1:2 to 3: 1, from 1:2 to 2:1, from 1:2 to 1: 1, from 1: 1 to 5: 1, from 1:1 to 4:1,from 1:1 to 3: 1, from 1:1 to 2: 1, from 2:1 to 5: 1, from 2:1 to 4: 1, from 2: 1 to 3:1, from 3: 1 to 5: 1, from 3:1 to 4: 1, or from 4: 1 to 5: 1.

[0139] In certain embodiments, the T 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 T cell therapy is allogeneic, optionally wherein the immune cells are genetically engineered to abrogate expression via component inactivation of class I MHC (e.g., 2M), class II (e.g., RFXANK), or TCR (e.g. TRAC, or CD3).

[0140] In certain embodiments, the engineered T cells express a chimeric receptor (e.g., a CAR) that specifically targets a cell expressing the targeted protein, e.g., a B cell expressing CD 19 or BMCA. In certain embodiments, the T cell is autologous to the subject. In other embodiments, the T cell is allogeneic to the subject.

[0141] 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 a[>TCRs or ybTCRs, and can be “trained” to express TCRs that target a given antigen or epitope. T cells can also be 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 T cells are 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.

[0142] The T cells for use in adoptive cell therapy can be engineered to express a CAR by introducing a heterologous polynucleotide encoding the CAR. In certain embodiments, the polynucleotide is a DNA molecule (e.g., a cDNA molecule). In certain embodiments, the polynucleotide further comprises an expression control sequence (e.g., promoter and / or enhancer) operably linked to the CAR coding sequence. In certain embodiments, the T cells are transduced by a vector, such as a viral vector (e.g., AAV vector, lentiviral vector, or adenoviral vector) or a non- viral vector (e.g., plasmid), comprising a polynucleotide encoding the CAR. In certain embodiments, the polynucleotide 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.

[0143] In certain embodiments, the therapeutic methods of the disclosure comprise administering a dose of engineered T cells (e.g., CAR T cells) to a subject in need thereof. Incertain embodiments, the dose of engineered T cells is from IxlO5cells / kg bodyweight to IxlO8cells / kg bodyweight. For example, in certain embodiments, the dose of engineered T cells is from IxlO5to IxlO8cells / kg, from IxlO5to 9xl07cells / kg, from IxlO5to 6xl07cells / kg, from IxlO5to 3xl07cells / kg, from IxlO5to IxlO7cells / kg, from IxlO5to 9x106cells / kg, from IxlO5to 8xl06cells / kg, from IxlO5to 7x106cells / 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 3x105cells / kg, from 3xl05to IxlO8cells / kg, from 3xl05to 9xl07cells / kg, from 3xlO5to 6xl07cells / kg, from 3xl05to 3xl07cells / kg, from 3xl05to IxlO7cells / kg, from 3xl05to 9xl06cells / kg, from 3xl05to 8xl06cells / kg, from 3xl05to 7xl06cells / kg, from 3xl05to 6xl06cells / kg, from 3xl05to 5xl06cells / kg, from 3xl05to 4xl06cells / kg, from 3xl05to 3x106cells / kg, from 3xl05to 2xl06cells / kg, from 3xl05to IxlO6cells / kg, from 3xlO5to 9xl05cells / kg, from 3xl05to 6xl05cells / kg, from 6xl05to IxlO8cells / kg, from 6xl05to 9xl07cells / kg, from 6xl05to 6xl07cells / kg, from 6xl05to 3xl07cells / kg, from 6xl05to IxlO7cells / kg, from 6xl05to 9xl06cells / kg, from 6xl05to 8xl06cells / kg, from 6xl05to 7x106cells / kg, from 6xl05to 6xl06cells / kg, from 6xl05to 5xl06cells / kg, from 6xl05to 4xl06cells / kg, from 6xl05to 3xl06cells / kg, from 6xl05to 2xl06cells / kg, from 6xl05to IxlO6cells / kg, from 6xl05to 9xl05cells / kg, from 9xl05to IxlO8cells / kg, from 9xl05to 9xl07cells / kg, from 9xl05to 6xl07cells / kg, from 9xl05to 3xl07cells / kg, from 9xl05to IxlO7cells / kg, from 9xl05to 9xl06cells / kg, from 9xl05to 8xl06cells / kg, from 9xl05to 7x106cells / kg, from 9x 105to 6x 106cells / kg, from 9xl05to 5xl06cells / kg, from 9xl05to 4xl06cells / kg, from 9xl05to 3xl06cells / kg, from 9xl05to 2xl06cells / kg, from 9xl05to IxlO6cells / 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 8x106cells / kg, from IxlO6to 7xl06cells / kg, from IxlO6to 6xl06cells / kg, from Ixl06to 5xl06cells / kg, from IxlO6to 4xl06cells / kg, from IxlO6to 3x106cells / kg, from Ixl06to 2xl06cells / kg, from 2xl06to IxlO8cells / kg, from 2xl06to 9xl07cells / kg, from 2xl06to 6xl07cells / kg, from 2xl06to 3xl07cells / kg, from 2xl06to IxlO7cells / kg, from 2xl06to 9xl06cells / kg, from 2xl06to 8xl06cells / kg, from 2xl06to 7x106cells / kg, from 2xl06to 6xl06cells / kg, from 2xl06to 5xl06cells / kg, from 2xl06to 4xl06cells / kg, from 2xl06to 3xl06cells / kg, from 3xl06to IxlO8cells / kg, from 3xl06to 9xl07cells / kg, from 3xl06to 6xl07cells / kg, from 3xl06to 3xl07cells / kg, from 3xl06to IxlO7cells / kg, from 3xl06to 9xl06cells / kg, from 3xl06to 8xl06cells / kg, from 3xl06to 7x106cells / kg, from 3xl06to 6xl06cells / kg, from 3xl06to 5xl06cells / kg, from 3xl06to 4xl06cells / kg, from 4xl06to IxlO8cells / kg, from 4xl06to 9xl07cells / kg, from 4xl06to 6xl07cells / kg, from 4xl06to 3xl07cells / kg, from 4xl06to IxlO7cells / kg, from 4xl06to 9xl06cells / kg, from 4xl06to 8xl06cells / kg, from 4xl06to 7xl06cells / kg, from 4xl06to 6xl06cells / kg, from 4xl06to 5xl06cells / kg, from 5xl06to IxlO8cells / kg, from 5xl06to 9xl07cells / kg, from 5xl06to 6xl07cells / kg, from 5xl06to 3xl07cells / kg, from 5xl06to IxlO7cells / kg, from 5xl06to 9xl06cells / kg, from 5xl06to 8xl06cells / kg, from 5xl06to 7xl06cells / kg, from 5xl06to 6xl06cells / kg, from 6xl06to IxlO8cells / kg, from 6xl06to 9xl07cells / kg, from 6xl06to 6xl07cells / kg, from 6xl06to 3xl07cells / kg, from 6xl06to IxlO7cells / kg, from 6xl06to 9xl06cells / kg, from 6xl06to 8xlOficells / kg, from 6xl06to 7xl06cells / kg, from 7xl06to IxlO8cells / kg, from 7xl06to 9xl07cells / kg, from 7xl06to 6xl07cells / kg, from 7xl06to 3xl07cells / kg, from 7xl06to IxlO7cells / kg, from 7xl06to 9xl06cells / kg, from 7xl06to 8xl06cells / kg, from 8xl06to IxlO8cells / kg, from 8xl06to 9xl07cells / kg, from 8xl06to 6xl07cells / kg, from 8xl06to 3xl07cells / kg, from 8xl06to IxlO7cells / kg, from 8xl06to 9xl06cells / kg, from 9xl06to IxlO8cells / kg, from 9xl06to 9xl07cells / kg, from 9xl06to 6xl07cells / kg, from 9xl06to 3xl07cells / kg, from 9xl06to IxlO7cells / kg, from IxlO7to 1x10scells / kg, from IxlO7to 9xl07cells / kg, from IxlO7to 6xl07cells / kg, from IxlO7to 3xl07cells / kg, from 3xl07to IxlO8cells / kg, from 3xl07to 9xl07cells / kg, from 3xl07to 6xl07cells / kg, from 6xl07to IxlO8cells / kg, from 6xl07to 9xl07cells / kg, or from 9xl07to IxlO8cells / kg. In certain embodiments, the dose of engineered T cells administered to the subject is from IxlO6to IxlO7cells / kg. In certain embodiments, the dose of engineered T cells is an effective amount of the engineered T cells.

[0144] In certain embodiments, the dose of engineered T cells is at least IxlO5cells / kg.For example, in certain embodiments, the dose of engineered T cells is at least IxlO8cells / kg, at least 9xl07cells / kg, at least 6xl07cells / kg, at least 3xl07cells / kg, at least IxlO7cells / kg, at least 9xl06cells / kg, at least 8xl06cells / kg, at least 7xl06cells / kg, at least 6xl06cells / kg, at least 5x106cells / kg, at least 4x106cells / kg, at least 3x106cells / kg, at least 2x106cells / kg, at least IxlO6cells / kg, at least 9xl05cells / kg, at least 6xl05cells / kg, at least 3xl05cells / kg, or at least IxlO5cells / kg. In certain embodiments, the dose of engineered T cells is at least IxlO6cells / kg. In certain embodiments, the dose of engineered T cells is IxlO8cells / kg, 9xl07cells / kg, 6xl07cells / kg, 3xl07cells / kg, IxlO7cells / kg, 9xl06cells / kg, 8xl06cells / kg, 7xl06cells / kg, 6xl06cells / kg, 5xl06cells / kg, 4xl06cells / kg, 3xl06cells / kg, 2xl06cells / kg, IxlO6cells / kg, 9xl05cells / kg, 6xl05cells / kg, 3xlO5cells / kg, or IxlO5cells / kg. In certainembodiments, the dose of engineered T cells is IxlO6cells / kg. In certain embodiments, the dose of engineered T cells is 3xl06cells / kg. In certain embodiments, the dose of engineered T cells is IxlO7cells / kg.

[0145] In certain embodiments, the total dose of engineered T cells (e.g., CAR T cells) administered to the subject is from IxlO6to IxlO10cells. For example, in certain embodiments, the total dose of engineered T cells administered to the subject is from IxlO6to IxlO10cells, from IxlO6to 5xl09cells, from IxlO6to IxlO9cells, from IxlO6to 5xlO8cells, from IxlO6to IxlO8cells, from IxlO6to 5xl07cells, from IxlO6to IxlO7cells, from IxlO6to 5xl06cells, from 5xl06to IxlO10cells, from 5xl06to 5xl09cells, from 5xl06to IxlO9cells, from 5xl06to 5xl08cells, from 5xl06to IxlO8cells, from 5xl06to 5xl07cells, from 5xl06to IxlO7cells, from IxlO7to IxlO10cells, from IxlO7to 5xl09cells, from IxlO7to IxlO9cells, from IxlO7to 5xl08cells, from IxlO7to IxlO8cells, from IxlO7to 5xl07cells, from 5xl07to IxlO10cells, from 5xl07to 5xl09cells, from 5xl07to IxlO9cells, from 5xl07to 5xl08cells, from 5xl07to 1x10scells, from IxlO8to IxlO10cells, from IxlO8to 5xl09cells, from 1x10sto IxlO9cells, from IxlO8to 5xl08cells, from 5xl08to IxlO10cells, from 5x10sto 5xl09cells, from 5xl08to IxlO9cells, from IxlO9to IxlO10cells, from IxlO9to 5xl09cells, or from 5xl09to IxlO10cells.

[0146] In certain embodiments, the total dose of engineered T cells e.g., CAR T cells) administered to the subject is at least IxlO6cells. For example, in certain embodiments, the total dose of engineered T cells administered to the subject is at least IxlO6cells, at least 2.5xl06cells, at least 5xl06cells, at least 7.5xl06cells, at least IxlO7cells, at least 2.5xl07cells, at least 5xl07cells, at least 7.5xl07cells, at least IxlO8cells, at least 2.5xl08cells, at least 5xl08cells, at least 7.5xl08cells, at least IxlO9cells, at least 2.5xl09cells, at least 5xl09cells, at least 7.5xl09cells, or at least IxlO10cells.

[0147] In certain embodiments, the engineered T cells are administered to the subject in a single dose. In certain embodiments, the engineered T cells are administered intravenously.

[0148] The disclosure further relates to pharmaceutical compositions comprising engineered T cells (e.g., CAR T cells) prepared using a method described herein, which may be used in the therapeutic methods of the disclosure. In certain embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier or excipient.IV. Therapeutic Uses

[0149] The present disclosure provides methods of decreasing an unwanted immune response in a subject by administering a genetically modified immune cell (e.g., a CAR T cell), wherein the T cell targets and kills a target immune cell (e.g., a B cell), thereby reducing the unwanted immune response in the subject.

[0150] 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 the immune 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 antibodyproducing 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.

[0151] Thus, the disclosure provides a method of treating an autoimmune disease in a subject. The method comprises administering to the subject engineered T cells generated by a method of the disclosure (e.g., CAR T cells), which target an immune cell, e.g., which target 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.

[0152] 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, 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, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, systemic sclerosis with renal involvement, systemic sclerosis with pulmonary involvement, systemic sclerosis with cardiac involvement, 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, juvenile myositis, systemic sclerosis, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, systemic sclerosis with renal involvement, systemic sclerosis with pulmonary involvement, systemic sclerosis with cardiac involvement, 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, 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, juvenile myositis, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, systemic sclerosis with renal involvement, systemic sclerosis with pulmonary involvement, systemic sclerosis with cardiac involvement, membranous nephropathy, anti-synthetase syndrome, dermatomyositis, or immune mediated necrotizing myopathy in a subject in need thereof.

[0153] 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.

[0154] 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.

[0155] 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 CD 19+ 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 CD19+ cells undetectable in the subject. In certain embodiments, the decreasein the amount of circulating CD 19+ cells is transient, and levels are restored partially or completely after a period of time.

[0156] In certain embodiments, therapeutic methods of the disclosure may be used to treat SLE in a subject.

[0157] The compositions and methods disclosed herein can also be used to treat various forms of cancer in a subject or inhibit cancer growth in a subject. The methods comprise administering to the subject an effective amount of a composition of engineered T cells prepared using a method described herein, either alone or in a combination with another therapeutic agent to treat the cancer in the subject.

[0158] Examples of cancers include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions. Examples of hematopoietic tumors include, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin’s disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, multiple myeloma, or Richter’s Syndrome (Richter’s Transformation). Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g., oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genitals and genitourinary tract (e.g., renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g., neural or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma).

[0159] In certain embodiments the cancer is an epithelial cancer. Exemplary epithelial cancers include, but are not limited to, endometrial cancer, colon, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer or fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary cancer, bladder cancer, head and neck cancer, oral cancer and liver cancer. Epithelial cancers also include carcinomas, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, baso squamous cellcarcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher’s carcinoma, Kulchitzky- cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0160] In certain embodiments, the cancer is an adenocarcinoma, a metastatic cancer, and / or is a refractory cancer. In certain embodiments, the cancer is a breast, colon or colorectal, lung, ovarian, pancreatic, prostate, cervical, endometrial, head and neck, liver, renal, skin, stomach, testicular, thyroid or urothelial cancer. In certain embodiments, the cancer is an epithelial cancer, e.g., an endometrial cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer, fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary cancer, bladder cancer, head and neck cancer, oral cancer or liver cancer.

[0161] In certain embodiments, the cancer is a hematologic cancer, e.g., a leukemia, lymphoma, or multiple myeloma, e.g., Chronic lymphocytic leukemia (CLL), Acute myeloid leukemia (AML), Chronic myelogenous leukemia (CML), Non-Hodgkin lymphoma (NHL), Burkitt lymphoma, Chronic myeloid monocytic leukemia (CMML), Eosinphilia, Essential thrombocytosis, Hairy cell leukemia, and NK cell lymphoma.

[0162] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. 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.

[0163] In certain embodiments, a method or composition described herein (e.g., a method of treating an autoimmune disease or a cell therapeutic composition for use in such a method) 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-proliferative compound, 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 oftherapeutics of different classes. In certain embodiments, the additional therapy may be an antibody therapy (e.g., belimumab) or a corticosteroid.

[0164] In certain embodiments wherein the disease to be treated is cancer, a method or composition described herein can administered in combination with one or more additional therapies such as surgery, radiation therapy, or administration of another therapeutic preparation. In certain embodiments, the additional therapy may include chemotherapy, e.g., a cytotoxic agent. In certain embodiments, the additional therapy may include a targeted therapy, e.g. a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In certain embodiments, the additional therapy may include an anti-inflammatory, anti- angiogenic, anti-fibrotic, or anti-proliferative compound, a steroid, a biologic immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodialator, 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.

[0165] In certain embodiments, a method or composition described herein is administered in combination with a checkpoint inhibitor. The checkpoint inhibitor may, for example, be selected from a PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, adenosine A2A receptor antagonist, B7-H3 antagonist, B7-H4 antagonist, BTLA antagonist, KIR antagonist, LAG3 antagonist, TIM-3 antagonist, VISTA antagonist or TIGIT antagonist.

[0166] In certain embodiments, the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. PD- 1 is a receptor present on the surface of T-cells that serves as an immune system checkpoint that inhibits or otherwise modulates T-cell activity at the appropriate time to prevent an overactive immune response. Cancer cells, however, can take advantage of this checkpoint by expressing ligands, for example, PD-L1, that interact with PD-1 on the surface of T-cells to shut down or modulate T-cell activity. Exemplary PD-1 / PD-L1 based immune checkpoint inhibitors include antibody based therapeutics. Exemplary treatment methods that employ PD-1 / PD-L1 based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994, and EP Patent No. 1537878B1, and, for example, include the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.),pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-Ll antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-Ll antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0167] In certain embodiments, a method or composition described herein is administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on a T-cell with its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of an antigen presenting cells (rather than cancer cells) leads to T-cell inhibition. Exemplary CTLA-4 based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International (PCT) Publication Nos. WO98 / 42752, WO00 / 37504, and WOOl / 14424, and European Patent No. EP 1212422 Bl. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0168] Exemplary cytotoxic agents that can be administered in combination with a method or composition described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinating agents, inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HD AC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkyl sulfonates, triazenes, folate analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis and radiation, or antibody molecule conjugates that bind surface proteins to deliver a toxic agent. In certain embodiment, the cytotoxic agent that can be administered with a method or composition described herein is a platinum-based agent (such as cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacytidine, vorinostat, ixabepilone, bortezomib, taxanes e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicin, anthracyclines (e.g., doxorubicin or epirubicin) daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids.V. Chimeric Antigen Receptors

[0169] Exemplary CARs that can be expressed by engineered T cells prepared using the 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.

[0170] In certain embodiments, a CAR of the disclosure comprises (1) an extracellular domain comprising an antigen-binding site that provides specificity for a desired antigen e.g., 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 targeted antigen, 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. WO2018 / 140725. a. CAR Extracellular Domain

[0171] 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, an autoantibody on the surface of an autoantibodyproducing B cell (in the form of a BCR).i. Antisen-binding Sites

[0172] 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.

[0173] 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 CD19, CD20, BCMA, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b or CD30. In certain embodiments, the antigen is CD 19.

[0174] 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.

[0175] Exemplary antigen-binding sites that bind CD 19 and that can be used in a CAR of the disclosure are described in greater detail hereinbelow.

[0176] 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 least96%, 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 antigenbinding site that binds CD19 comprises CDRHI, CDRH2, CDRHS, CDRLI, CDRL2, and CDRL3 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 CD19 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, CDRHI, 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 CDRm, CDR112, CDRHS, CDRLI, CDRL2, and CDRL3 sequences determined under Kabat (see Kabat et 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, CDRH3, CDRLI, CDRL2, and CDRL3 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.).

[0177] 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. 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)

[0178] In certain embodiments, the antigen-binding site comprises the CDRHI, CDRH2, CDRHB, CDRLI, CDRL2, and CDRL3 sequences of an antigen-binding site disclosed in TABLE 2, where the CDR sequences in each of the VH and VL sequences are underlined. 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)

[0179] 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 3, where the CDR sequences in each of the VH and VL sequences are underlined. 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)

[0180] In certain embodiments, the antigen-binding site that binds CD 19 comprises a Vn comprising CDRm, CDRm, and CDRm sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, 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 in 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 CDRm, CDRm, and CDRm sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0181] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRm, CDRm, and CDRm sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, 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 in 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 CDRm, CDRm, and CDRm sequences and / or the CDRLI , CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0182] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth in SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively, wherein CDRHI, CDRH2, and CDRH3 sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRm sequences set forth in 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 CDRH3 sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0183] In certain embodiments, the antigen-binding site that binds CD19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 30, 31, 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 in 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.

[0184] In certain embodiments, the antigen-binding site that binds CD19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 36, 37, 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 in SEQ ID NOs: 39, 40, and 16, respectively, wherein CDRLI, CDRL2, and CDRT.3 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.

[0185] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 108, 109, and 43, respectively, wherein CDRHI, CDRH2, and CDRH sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively, wherein CDRLI, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR sequences. In certainembodiments, the CDRHI, CDRH2, and CDRH3 sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0186] In certain embodiments, the antigen-binding site that binds CD19 comprises a VH comprising CDRHI, CDRH2, and CDRH3 sequences set forth in SEQ ID NOs: 1 10, 11 1 , and 32, respectively, wherein CDRm , CDR112, and CDRIB sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth in 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, CDRm, and CDRm sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0187] In certain embodiments, the antigen-binding site that binds CD 19 comprises a VH comprising CDRHI, CDRm, and CDRm sequences set forth in SEQ ID NOs: 112, 113, and 38, respectively, wherein CDRHI, CDRm, and CDRm sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth in 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, CDRm, and CDRm sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0188] In certain embodiments, the antigen-binding site that binds CD19 comprises a VH comprising CDRHI, CDRm, and CDRm sequences set forth in SEQ ID NOs: 41, 42, and 43, respectively, wherein CDRHI, CDRm, and CDRm sequences are interposed between immunoglobulin FR sequences; and / or a VL comprising CDRLI, CDRL2, and CDRL3 sequences set forth in 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, CDRm, and CDRm sequences and / or the CDRLI, CDRL2, and CDRL3 sequences are interposed between human or humanized immunoglobulin FR sequences.

[0189] In certain embodiments, the antigen-binding site that binds CD19 comprises a VH comprising the amino acid sequence of SEQ ID NO: 4, 13, or 44. In certain embodiments,the antigen-binding site that binds CD 19 comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, 17, or 48.

[0190] In certain embodiments, the antigen-binding site that binds CD 19 comprises a Vn comprising the amino acid sequence of SEQ ID NO: 4, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0191] In certain embodiments, the antigen-binding site that binds CD19 comprises a Vn comprising the amino acid sequence of SEQ ID NO: 13, and / or a VL comprising the amino acid sequence of SEQ ID NO: 17.

[0192] In certain embodiments, the antigen-binding site that binds CD19 comprises a Vn comprising the amino acid sequence of SEQ ID NO: 44, and / or a VL comprising the amino acid sequence of SEQ ID NO: 48.

[0193] In certain embodiments, an antigen-binding site that binds CD19 comprises a Vn 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 CD 19 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.

[0194] In certain embodiments, an antigen-binding site that binds CD 19 comprises a Vn 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 least94.5%, 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.

[0195] In certain embodiments, an antigen-binding site that binds CD19 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.

[0196] In certain embodiments, an antigen-binding site that binds CD19 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.

[0197] 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 of the VH are humanized or human framework regions. In certain embodiments, the framework regions of the VL are humanized or human framework regions.

[0198] In certain embodiments, an antigen-binding site that binds CD19 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.

[0199] In certain embodiments, an antigen-binding site that binds CD19 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.

[0200] 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.

[0201] 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.

[0202] 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 CD19 is fully human.

[0203] 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; and2021 / 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.

[0204] 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 antigen-binding 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.

[0205] In certain embodiments, the antigen-binding site that binds CD19 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.

[0206] 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-specificinteractions, 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.

[0207] 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.

[0208] 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.

[0209] 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

[0210] 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 may comprise 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.

[0211] 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.

[0212] 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.

[0213] 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 position corresponding to position 96 of wild-type human MuSK is substituted with alanine (e.g., as in SEQ ID NO: 55).

[0214] 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.

[0215] 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.

[0216] 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 certain embodiments, the AChR autoantigen comprises the amino acid sequence of any one of SEQ ID NOs: 60-67.

[0217] 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, 1 1, 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.

[0218] 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 PLA2Rautoantigen 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 least99%) 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.

[0219] 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.

[0220] 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.

[0221] 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 than14, 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, 1 1, 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

[0222] 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 chain, TCR chain, CD28, CD3s, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, EGFR, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R , IL2Ry, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, 1TGAX, CDl lc, 1TGB 1, CD29, 1TGB2, CD18, LFA-1, 1TGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), 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, thetransmembrane 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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 certainembodiments, 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.

[0227] 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 ( 19ql 3.4). 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 DAP12. In certain embodiments, the KIR is KIRS2 or KIR2DS2. c. CAR Intracellular Domains

[0228] 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.

[0229] Intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs). Examples of ITAM-containing cytoplasmic signaling sequences that are ofparticular use in the CARs of the present application include those derived from CD3^, common FcRy (FCER1G), FcyRIIa, FcR(3 (FceRlb), CD3y, CD35, 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, FcR|3, CD3y, CD35, CD3e, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, common FcRy (FCER1G), FcyRIIa, FcR0 (FceRlb), DAP10, and / or DAP12. In certain embodiments, the intracellular signaling domain comprises a CD3 signaling domain or an FcRy signaling domain.

[0230] In certain embodiments, a CAR of the disclosure comprises a CD3c signaling domain by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the CAR can comprise a CD3 chain portion (z.e., a CD3 intracellular signaling domain) and an intracellular domain of a costimulatory molecule, for example, a 4- IBB intracellular domain or a CD28 intracellular 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.

[0231] In certain embodiments, a CAR of the disclosure comprises a CD3C 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.

[0232] 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.

[0233] 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, CD1 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, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB- A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), 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 (CDl 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- IBB intracellular domain and / or a CD28 intracellular domain.

[0234] 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.

[0235] In certain embodiments, the costimulatory domain comprises a conservative substitution relative to a costimulatory domain disclosed herein, e.g., relative to costimulatorydomain 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.

[0236] 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.

[0237] 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.

[0238] 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)n linker. In certain embodiments, the intracellular signaling domain (e.g. CD3 signaling domain) is the C-terminal domain of the CAR.d. Other CAR Domains

[0239] 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. A variety 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 (G4S)4 linker, a KIR2DS2 hinge, and a CD8a hinge.

[0240] 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.

[0241] 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.

[0242] In certain embodiments, the CAR comprises a CD8a transmembrane domain and a CD8a hinge.

[0243] 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.

[0244] 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 of SEQ 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.

[0245] 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

[0246] 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- IBB, 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 2A-2B.

[0247] 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.

[0248] 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.

[0249] 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 least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 28 or SEQ IDNO: 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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, 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%, atleast 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.

[0254] 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 substitutions relative 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 certainembodiments, 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

[0255] In certain embodiments, engineered T cells produced using the methods of the disclosure may express a CAR encoded by a polynucleotide 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- IBB costimulatory domain, and a CD3^ signaling domain.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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 alsohave the added advantage of resulting in low immunogenicity in the subject into which they are introduced.

[0260] In 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.

[0261] 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.

[0262] 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).

[0263] 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 of the 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.

[0264] An example of a promoter that may be used is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promotersequence 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- 1 a 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.

[0265] 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 non-limiting 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 in molecular circuits results in a logic gate. See, for example, Roybal et al., 2016 CELL 164(4): 770-9.

[0266] Examples of other systems for expressing or regulating expression of a chimeric receptor include those described in Wu et al. (2015) Science 350: aab4077; Fedorov et al. (2014) Cancer lournal 20:160-165; Kloss et al. (2013) Nature Biotechnology 31 : 71-75; Sakemura et al. (2016) Cancer Immunol. Res. 4:658-668; Hill et al. (2018) NAT. CIEM. BIOL. 14: 112-117; Di Stasi et al. (2011) N. ENGL. J. MED. 365: 1673-1683; Budde er aZ. (2013) PLoS ONE 8: e82742; Wei et al. (2012) NATURE 488: 384-388; Ma et al. (2016) PROC. NATL. ACAD. Set. USA 113: E450-458; Rodgers et al. (2016) PROC. NATL. ACAD. Set. USA 113:E459-468; Kudo et al. (2014) CANCER RES. 74: 93-103, and Chen et al. (2010) PROC. NATL.ACAD. Set. USA 107: 8531-8536.

[0267] 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.

[0268] 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, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 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 be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0269] 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.

[0270] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acidsare 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).

[0271] 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.

[0272] 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 sub-micron sized delivery system.

[0273] 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 a liposome, 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, aminoalcohols, 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.

[0274] 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 et al. (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.

[0275] 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.

[0276] 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.VI. Pharmaceutical Compositions

[0277] As described hereinabove, the engineered T cells of the disclosure (e.g., CAR-T cells) 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 engineered T cells produced using the methods described herein. In certain embodiments, the engineered T cells are present in a pharmaceutical composition, e.g., wherein the composition comprises a pharmaceutically acceptable carrier or excipient.

[0278] In certain embodiments, the T cell therapy is autologous, i.e., T 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 T cells are obtained from a healthy donor, optionally wherein the T cells (e.g., CAR-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 orthe TCR P 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 ( 2M). 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. In embodiments, gene editing systems targeting sequences encoding TCR a chain, TCR chain, P2M, 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..

[0279] A T cell therapy can be provided as a cell composition, e.g., a CAR-T cell composition generated by a method of the disclosure. 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%, at least 99%, or at least 99.5% 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, 1% or less, or 0.5% or less.

[0280] In certain embodiments, an immune cell composition may comprise one or more immunogenicity enhancing adjuvants (also referred to as “adjuvants” herein). Such adjuvantsare 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®, AS15, 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 -|3, 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 et al. (1995) PllARM. BIOTECIINOL. 6: 277-96). In certain embodiments, an adjuvant is a naturally occurring adjuvant. In certain embodiments, an adjuvant is a non-naturally occurring adjuvant.

[0281] The therapeutically effective amount of the engineered T cells 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.

[0282] 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 presentinvention 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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

[0288] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1: Small- and large-scale manufacturing of CAR T cells from Whole Blood

[0289] This example describes a process for manufacturing CAR T cells using whole blood as a source of lymphocytes, as summarized in the schematic of FIGURE 1. Current clinical manufacturing processes require cells from a leukapheresis source as the starting material, and the reliance on apheresis collection creates a bottleneck that reduces efficiency and accessibility of CAR-T cell therapy. The use of whole blood as starting material would resolve this bottleneck.

[0290] Preparations of CD 19 CAR T cell were made by processing 80-200 mL of whole blood collected from patients. Whole blood or leukapheresis product was collected. Collected whole blood was delivered directly to the preparation facility and held overnight at ambient temperature for less than or up to 24 hours, less than or up to 48 hours, or less than or up to 72 hours before processing was initiated. PBMCs were isolated from a fresh whole blood collection via density gradient centrifugation using the NeatCell program on Sepax C- Pro, and the resulting PBMCs were cryopreserved. Prior to initiating CAR T cell preparation (Day 0), the cryopreserved PBMCs were thawed and assessed for the percentage of CD3+ cells via flow cytometry. All available PBMCs were enriched for CD4+ and CD8+ cells using the TCT application on the CliniMACS Prodigy, and the enriched fraction was used for CAR T generation.

[0291] For small-scale manufacturing, on Day 0, the previously enriched cells were seeded into multiple 6M G-Rex wells at seeding densities ranging from 5E+5 to 1E+6 cells / cm2and activated with RUO grade TransAct at a volume:volume ratio of 4:70 TransAct to culture medium. The culture was seeded in a total of 10 mL with TexMACS + 12.5 ng / mL of IL-7 and IL-15. On Day 1, lentiviral vector encoding a CD19-CAR (comprising a CD8 transmembrane domain, a 4 IBB intracellular domain, a CD3^ signaling domain, and an extracellular anti-CD19 antigen binding site corresponding to “CD19-1” in TABLES 1-3) was added at an MOI of 8. On Day 3, the culture was washed and resuspended in TexMACS + 12.5 ng / mL of recombinant IL-7 and recombinant IL-15 + 5% Human AB Serum, then transferred back into the respective 6M G-Rex well in a total volume of 100 mL. Ifappropriate, cells were harvested from individual wells on Days 3 and 6 and cryopreserved to capture intermediate timepoints. At least one well was left to expand through Day 9, at which point cells were harvested and cryopreserved for further testing.

[0292] For large-scale manufacturing, on Day 0, the previously enriched cells were seeded into the culture chamber of the CliniMACS Prodigy (up to a max of lE+8 viable cells) and activated with GMP grade TransAct at a volume:volume ratio of 4:70 TransAct to culture medium. The culture was seeded in a total of 70 mL with TexMACS + 12.5 ng / mL of IL-7 and IL-15. On Day 1, lentiviral vector encoding a CD19-CAR (comprising a CD8 transmembrane domain, a 41BB intracellular domain, a CD3 signaling domain, and an extracellular anti-CD19 antigen binding site corresponding to “CD19-1” in TABLES 1-3) was added at an MOI of 8. On Day 3, the culture was washed and resuspended in TexMACS + 12.5 ng / mL of recombinant IL-7 and recombinant IL- 15 + 5% Human AB Serum, then transferred into a 100M G-Rex in a total volume of 1 L. The culture was left to expand through Day 9, at which point cells were harvested and washed using the CultureWash program on the Sepax C-Pro. Harvested and washed cells were cryopreserved for further testing. Day 6 samples were only collected in the small scale studies.Example 2; Comparison of CAR T cells manufactured from whole blood samples and leukapheresis samples

[0293] This example describes experiments validating the growth and functionality of CAR T cells manufactured from a whole blood collection source.

[0294] Whole blood was processed at a small scale as described in Example 1 or through standard leukapheresis-based processes from three matched healthy human donors (HD1, HD2, HD3). Cell growth was evaluated over time during the manufacturing process by measuring viable nucleated cells (VNCs; FIGURE 3). Cells from all donors grew similarly, irrespective of whether a whole blood sample or a leukapheresis sample was used as the starting material.

[0295] At each culturing timepoint, at least 2E5 cells per condition were collected, resuspended in flow buffer, and fluorescently labeled with the following reagent per manufacturer instructions: PacificBlue CD4, BV605 CD279 (PD1), BV650 CD45RA, PerCP / Cy5.5 CD3, PE / Cy7 CD366 (Tim-3), APC CD197 (CCR7), APC / Cy7 CD8, PE- Vio710 CD25, FITC CD69 (Biolegend), PE-labeled CD19 protein His Tag detection reagent(ACRO Biosystems), and viobility 405 / 520 fixable dye (Miltenyi Biotec). Following labeling, cells were fixed using BD Cytofix™ fixation buffer and analyzed using an Agilent Novocyte Quanteon flow cytometer. FIGURE 4 shows an exemplary plot of cells isolated from healthy donor HD1. Expression of the CD19-CAR in the prepared populations of engineered cells was measured by flow cytometry. As shown in FIGURE 4 for healthy donor HD1, the percentage of CAR 19+ CD3 cells in the population of cells prepared from a leukapheresis sample was 75.79%, and the percentage of CAR19+ CD3 cells in the population of cells prepared from a whole blood sample was 64.88%. The percentage of CD19-CAR+ CD3 cells in the Day 9 populations, the fold expansion of cells from Day 0 to Day 9, and the number of CD19-CAR+ cells in the Day 9 populations are summarized in TABLE 4 for leukapheresis-derived and whole-blood-derived samples from all three donors. The percentages of Tn, Temra, Tem, and Tcm cells in the Day 9 populations of CD19-CAR+ CD3 cells were analyzed by detecting CCR7 and CD45RA expression by flow cytometry. As shown in FIGURES 5A-5B, the CAR-T cell populations were found to have comparable memory phenotypes, irrespective of whether the cells were prepared from leukapheresis or whole blood samples. Expression of T-cell activation markers (CD25 and CD69) and T-cell exhaustion markers (PD1 and TIM3) in the CAR T cell populations was also measured by flow cytometry. As shown in FIGURE 6, expression of the activation and exhaustion markers were comparable between cells produced from whole blood samples and leukapheresis samples.TABLE 4.

[0296] Proliferation of the prepared CAR T cells was also measured during co-culture with CD19-expressing target cells (Nalm6 cells). Briefly, CAR-T cells were cultured with Nalm6 cells at an effector to target (E:T) ratio of 1 : 1 (“stimulated”). CAR-T cells that were not cultured with Nalm6 cells were used as a control (“unstimulated”). Approximately 1 E+6 CAR T cells were plated in 2 mL complete RPMI media with 10% FBS (cRIO) in a cell culture treated 6-well plate. Approximately 1E+06 Nalm6 cells in 2 mL cRIO were added to each culture, for a total volume of 4 mL. Cells were kept in an incubator at 37°C (5% CO2) and counted every 3-4 days via a cell counter, using Viastain AO / PI staining solution. Following each count, cells were re-plated at 0.8E+06 cells / mL concentration, in a culturing vessel with an area density around 0.2~0.5E+06 cells / cm2to continue culturing. In addition, an identical culture plate with the same co-culture conditions was used to determine the T- cell phenotype for each culture using flow cytometry as described above. The percentage of CD3+ and CD 19 CAR+ cells was then used to estimate a growth curve of each co-culture. T-cell proliferation was followed for 14 days after initial target cell stimulation for CAR-T cells prepared from leukapheresis- and whole-blood-derived samples from all three donors. No statistical differences were observed between whole blood or leukapheresis derived T- cells at any time point (FIGURE 7).

[0297] The cytotoxic activity of CD 19+ CAR T cells prepared from whole blood or leukapheresis samples was measured for all three donors. To measure activity, the ability of CD19-CAR T cells to kill CD19-expressing target cells (Nalm6 cells) was evaluated using an IncuCyte assay. Briefly, Nalm6 cells endogenously expressing CD19 and modified to express GFP were co-cultured with CAR-T cells or non-transduced (NTD) T cells at Effector to Target (E:T) ratios of 1 : 1, 0.5:1, 0.25:1 , 0.125:1, and 0.0625:1. E:T ratios were adjusted based on the expression level of CD19-CAR to match the transduced T cell to target cell ratios. Co-cultures were monitored for 120 hours using IncuCyte. Images were taken every hour with a lOx objective. GFP-positive cells were quantified as the mean number of cell counts in each imaging field. Dying or dead Nalm6 lose the GFP+ signature and are not included in the GFP+ cell counts. FIGURE 8A and FIGURE 8B summarize the cytotoxic activity of CAR T cells when co-cultured with target cells at an 1: 1 E:T ratio and a 0.125:1 E:T ratio, respectively. The area under the curve (AUC) was also calculated for each E:T ratio tested. As shown in FIGURE 8C, CAR T cells prepared from whole blood or bystandard leukapheresis processing demonstrated comparable, effective killing of CD 19+ target cells.

[0298] Populations of CAR T cells were generated using 200 mb of whole blood from two healthy human donors as starting material, using the large-scale manufacturing protocol described in Example 1 . Leukapheresis process controls were from un-matched donors. The growth and activity of CAR T cells were measured using the same assays used to analyze the CAR-T cells produced using the small-scale protocol. Measuring the counts of viable nucleated cells (VNCs) during the course of manufacturing showed that CAR T cells from either the large-scale whole blood preparation or standard leukapheresis preparation grew similarly (FIGURE 9A). CAR T cells manufactured from whole blood from both human donors showed efficient target-cell-killing at E:T ratios of 1:1, 0.5:1, 0.25:1, 0.125:1, and 0.0625:1 (FIGURE 9B). The percentage of CD19-CAR+ CD3 cells in the Day 9 populations, the fold expansion of cells from Day 0 to Day 9, and the number of CD19- CAR+ cells in the Day 9 populations are summarized in TABLE 5 for leukapheresis-derived and whole-blood-derived samples.TABLE 5Example 3: CAR T cells from whole blood are effective in treatment of an autoimmune disease

[0299] This example describes use of CD19-CAR T cells manufactured from a whole blood collection source from human donors with systemic lupus erythematosus SEE. CAR T cell therapy shows promise in treatment for autoimmune diseases, such as B-cell-mediatedautoimmune diseases including refractory systemic lupus erythematosus (SLE).Accordingly, it is useful to generate CAR T cells derived from these patients using whole blood as a starting product.

[0300] Whole blood from the two SLE donors was processed at a small scale as described in Example 1 and validated for CD19 CAR expression and cytotoxicity using the methods described in Example 2. The percentage of CD19-CAR+ T cells as determined by flow cytometry was 46.99% (FIGURE 10). The SLE donor CAR T cells were co-cultured with Nalm6 target cells at Effector to Target ratios of 1: 1, 0.5: 1, 0.25: 1, 0.125:1, and 0.0625: 1, and showed effective killing at all tested ratios (FIGURE 11). T cells populations displayed expected sub-populations of memory T cells (Tn, Temra, Tern, and Tcm cells), as shown in an exemplary flow cytometry scatter plot for one donor (FIGURE 12A) and in a bar plot summarizing the results for both donors (FIGURE 12B). Activation and exhaustion of T cells from both donors was detected by flow cytometry for CD25 and CD69 markers and PD- 1 and TIM-3 markers, respectively (FIGURE 13). Together, these data show that functional CD 19 CAR T cells can be effectively manufactured from whole blood as a starting material in the context of individuals suffering from the autoimmune disease systemic lupus erythematosus (SLE).INCORPORATION BY REFERENCE

[0301] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0302] 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 and range of equivalency of the claims are intended to be embraced therein.SEQUENCE LISTING

Claims

1. WHAT IS CLAIMED IS:

1. A method of producing a composition of engineered T cells that express a chimeric receptor, the method comprising:(a) contacting a population of primary T cells with an activating agent, wherein the population of primary T cells has been obtained directly from a whole hlood sample isolated from a subject, thereby generating a population of activated T cells;(b) introducing into the activated T cells a heterologous polynucleotide encoding the chimeric receptor, thereby generating a population of genetically modified T cells;(c) incubating the population of genetically modified T cells in a basal culture medium for at least two days, thereby generating a population of expanded T cells; and(d) harvesting the expanded T cells, thereby producing a composition of engineered T cells.

2. The method of claim 1, wherein the whole blood sample obtained from the subject comprises 50-300 mL of whole blood.

3. The method of claim 1 or 2, wherein the whole blood sample obtained from the subject comprises 50-250 mL of whole blood.

4. The method of any one of claims 1-3, wherein the whole blood sample obtained from the subject comprises 80-250 mL of whole blood.

5. The method of any one of claims 1-4, wherein PBMCs are isolated from the whole blood sample using density gradient centrifugation.

6. The method of any one of claims 1-4, wherein PBMCs are isolated from the whole blood sample by adding a red-blood-cell lytic agent to the whole blood sample.

7. The method of claim 6, wherein the red-blood-cell lytic agent is selected from the group consisting of water and ammonium-chloride-potassium (ACK) lysis buffer.

8. The method of any one of claims 1-7, wherein the subject has an autoimmune disease.

9. The method of claim 8, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myastheniagravis (MG), myositis, systemic sclerosis, multiple sclerosis, membranous nephropathy, and chronic immune demyelinating polyneuropathy.

10. The method of claim 9, wherein the autoimmune disease is SLE.

11. The method of claim 9, wherein the autoimmune disease is PV.

12. The method of claim 9, wherein the autoimmune disease is MG.

13. The method of claim 9, wherein the autoimmune disease is myositis.

14. The method of claim 9, wherein the autoimmune disease is systemic sclerosis.

15. The method of claim 9, wherein the autoimmune disease is multiple sclerosis.

16. The method of claim 9, wherein the autoimmune disease is membranous nephropathy.

17. The method of claim 9, wherein the autoimmune disease is chronic immune demyelinating polyneuropathy.

18. The method of any one of claims 1-17, wherein the population of primary T cells is contacted with the activating agent within 24, 48, or 72 hours of when the whole blood sample is isolated from the subject.

19. The method of any one of claims 1-18, wherein the method further comprises, prior to step (a):(I) obtaining a population of PBMCs isolated from the whole blood sample; and(II) enriching for CD4+ and CD8+ T cells in the population of PBMCs, thereby generating the population of primary T cells.

20. The method of claim 19, wherein step (I) comprises thawing a frozen sample comprising the population of PBMCs.

21. The method of claim 19, wherein the population of PBMCs is never frozen.

22. The method of claim 21 , wherein the population of PBMCs is stored at a temperature above 0 °C and below 22 °C.

23. The method of any one of claims 19-22, wherein step (II) comprises positively selecting for CD4+ T cells and CD8+ T cells.

24. The method of any one of claims 19-23, wherein the population of primary T cells is frozen after being generated in step (II).

25. The method of any one of claims 1-18, wherein the method further comprises, prior to step (a), a step of (I) enriching for CD4+ and CD8+ T cells in the whole blood sample, thereby generating the population of primary T cells.

26. The method of claim 25, wherein the population of primary T cells is frozen after being generated in step (I).

27. The method of any one of claims 1-19 or 25, wherein the whole blood sample and / or the population of primary T cells is never frozen.

28. The method of claim 27, wherein the whole blood sample and / or the population of primary T cells is stored at a temperature above 0 °C and below 22 °C.

29. The method of any one of claims 5-7, wherein the PBMCs are frozen after being isolated from the whole blood sample.

30. The method of any one of claims 1 -29, wherein the activating agent comprises an antibody or an antigen-binding fragment thereof.

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

32. The method of claim 30 or 31 , wherein the activating agent comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

33. The method of any one of claims 1-32, wherein the activating agent is bound to a bead.

34. The method of claim 33, wherein the bead is a magnetic bead.

35. The method of any one of claims 1-32, wherein the activating agent is bound to a polymeric nanomatrix.

36. The method of any one of claims 1-35, wherein the contacting in step (a) is carried out for at least 12 hours.

37. The method of any one of claims 1-36, wherein the contacting in step (a) is carried out for at least 24 hours.

38. The method of any one of claims 1-37, wherein step (a) and / or step (b) is carried out in a further basal culture medium.

39. The method of claim 38, wherein step (a) comprises seeding the population of primary T cells in the further basal culture medium at a concentration of 5xl05to 5xl06cells per mL.

40. The method of claim 38 or 39, wherein the further basal culture medium comprises IL-2, IL-7, and / or IL- 15.

41. The method of any one of claims 38-40, wherein the further basal culture medium comprises IL-7 and IL- 15.

42. The method of any one of claims 38-41, wherein step (b) comprises culturing the activated T cells in the further basal culture medium for at least 1 day or at least 2 days.

43. The method of any one of claims 1-42, wherein heterologous polynucleotide is included in a viral vector.

44. The method of claim 43, wherein the viral vector is a lentiviral vector or an adeno- associated viral vector.

45. The method of claim 44, wherein the viral vector is a lentiviral vector.

46. The method of any one of claims 43-45, wherein the population of activated T cells is incubated with the viral vector at a multiplicity of infection (MOI) of 4 to 10.

47. The method of any one of claims 43-46, wherein the population of activated T cells is incubated with the viral vector at an MOI of 7 to 9.

48. The method of any one of claims 1-47, wherein the basal culture medium comprises IL-2, IL-7, and / or IL-15.

49. The method of claim 48, wherein the basal culture medium comprises IL-7 and IL- 15.

50. The method of any one of claims 1 -49, wherein the basal culture medium comprises human male AB serum (HABS).

51. The method of any one of claims 1-50, wherein step (c) is carried out in a bioreactor.

52. The method of any one of claims 1-51, wherein step (c) comprises incubating the population of genetically modified T cells in a basal culture medium for at least three days, at least four days, at least five days, or at least six days.

53. The method of any one of claims 1-52, wherein the whole blood sample has not been subjected to leukapheresis.

54. A method of producing a composition of engineered T cells that express a chimeric receptor, the method comprising:(a) contacting a population of primary T cells with an activating agent, wherein the population of primary T cells has been obtained from a sample isolated from a subject, thereby generating a population of activated T cells;(b) introducing into the activated T cells a heterologous polynucleotide encoding the chimeric receptor, thereby generating a population of genetically modified T cells;(c) incubating the population of genetically modified T cells in a basal culture medium for at least two days, thereby generating a population of expanded T cells; and(d) harvesting the expanded T cells, thereby producing a composition of engineered T cells, wherein:(i) the subject has an autoimmune disease; and / or(ii) the population of primary T cells is contacted with the activating agent within 72 hours of when the sample was isolated from the subject.

55. The method of claim 54, wherein the sample is a whole blood sample.

56. The method of claim 55, wherein the whole blood sample obtained from the subject comprises 50-300 mL of whole blood.

57. The method of claim 55 or 56, wherein the whole blood sample obtained from the subject comprises 50-250 mL of whole blood.

58. The method of any one of claims 55-57, wherein the whole blood sample obtained from the subject comprises 80-250 mL of whole blood.

59. The method of any one of claims 55-58, wherein PBMCs are isolated from the whole blood sample using density gradient centrifugation.

60. The method of any one of claims 55-58, wherein PBMCs are isolated from the whole blood sample by adding a red-blood-cell lytic agent to the whole blood sample.

61. The method of claim 60, wherein the red-blood-cell lytic agent is selected from the group consisting of water and ammonium-chloride-potassium (ACK) lysis buffer.

62. The method of any one of claims 54-61, 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.

63. The method of claim 62, wherein the autoimmune disease is SLE.

64. The method of claim 62, wherein the autoimmune disease is PV.

65. The method of claim 62, wherein the autoimmune disease is MG.

66. The method of claim 62, wherein the autoimmune disease is myositis.

67. The method of claim 62, wherein the autoimmune disease is systemic sclerosis.

68. The method of claim 62, wherein the autoimmune disease is multiple sclerosis.

69. The method of claim 62, wherein the autoimmune disease is membranous nephropathy.

70. The method of claim 62, wherein the autoimmune disease is chronic immune demyelinating polyneuropathy.

71. The method of any one of claims 54-70, wherein the population of primary T cells is contacted with the activating agent within 24, 48, or 72 hours of when the sample is isolated from the subject.

72. The method of any one of claims 54-71, wherein the method further comprises, prior to step (a):(I) obtaining a population of PBMCs isolated from the sample; and(II) enriching for CD4+ and CD8+ T cells in the population of PBMCs, thereby generating the population of primary T cells.

73. The method of claim 72, wherein step (1) comprises thawing a frozen sample comprising the population of PBMCs.

74. The method of claim 72, wherein the population of PBMCs is never frozen.

75. The method of claim 74, wherein the population of PBMCs is stored at a temperature above 0 °C and below 22 °C.

76. The method of claim 72, wherein step (II) comprises positively selecting for CD4+ T cells and CD8+ T cells.

77. The method of any one of claims 72-76, wherein the population of primary T cells is frozen after being generated in step (11).

78. The method of any one of claims 55-61, wherein the method further comprises, prior to step (a), a step of (I) enriching for CD4+ and CD8+ T cells in the whole blood sample, thereby generating the population of primary T cells.

79. The method of claim 78, wherein the population of primary T cells is frozen after being generated in step (I).

80. The method of any one of claims 55-61 or 78, wherein the whole blood sample and / or the population of primary T cells is never frozen.

81. The method of claim 80, wherein the whole blood sample and / or the population of primary T cells is stored at a temperature above 0 °C and below 22 °C.

82. The method of any one of claims 59-61, wherein the PBMCs are frozen after being isolated from the whole blood sample.

83. The method of any one of claims 54-82, wherein the activating agent comprises an antibody or an antigen-binding fragment thereof.

84. The method of claim 83, wherein the antibody or antigen-binding fragment thereof is humanized or fully human.

85. The method of claim 83 or 84, wherein the activating agent comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

86. The method of any one of claims 54-85, wherein the activating agent is bound to a bead.

87. The method of claim 86, wherein the bead is a magnetic bead.

88. The method of any one of claims 54-85, wherein the activating agent is bound to a polymeric nanomatrix.

89. The method of any one of claims 54-88, wherein the contacting in step (a) is carried out for at least 12 hours.

90. The method of any one of claims 54-89, wherein the contacting in step (a) is carried out for at least 24 hours.

91. The method of any one of claims 54-90, wherein step (a) and / or step (b) is carried out in a further basal culture medium.

92. The method of claim 91 , wherein step (a) comprises seeding the population of primary T cells in the further basal culture medium at a concentration of 5xl05to 5xl06cells per mL.

93. The method of claim 91 or 92, wherein the further basal culture medium comprises IL-2, IL-7, and / or IL- 15.

94. The method of any one of claims 91-93, wherein the further basal culture medium comprises IL-7 and IL- 15.

95. The method of any one of claims 91-94, wherein step (b) comprises culturing the activated T cells in the further basal culture medium for at least 1 day or at least 2 days.

96. The method of any one of claims 54-95, wherein heterologous polynucleotide is included in a viral vector.

97. The method of claim 96, wherein the viral vector is a lentiviral vector or an adeno- associated viral vector.

98. The method of claim 97, wherein the viral vector is a lentiviral vector.

99. The method of any one of claims 96-98, wherein the population of activated T cells is incubated with the viral vector at a multiplicity of infection (MOI) of 4 to 10.

100. The method of any one of claims 96-99, wherein the population of activated T cells is incubated with the viral vector at an MOI of 7 to 9.

101. The method of any one of claims 54-100, wherein the basal culture medium comprises IL-2, IL-7, and / or IL- 15.

102. The method of any one of claims 54-101, wherein the basal culture medium comprises IL-7 and IL- 15.

103. The method of any one of claims 54-102, wherein the basal culture medium comprises human male AB serum (HABS).

104. The method of any one of claims 54-103, wherein step (c) is carried out in a bioreactor.

105. The method of any one of claims 54-104, wherein step (c) comprises incubating the population of genetically modified T cells in the basal culture medium for at least three days, at least four days, at least five days, or at least six days.

106. The method of any one of claims 54-105, wherein the sample has not been subjected to leukapheresis.

107. The method of any one of claims 1-106, wherein at least 50%, at least 55%, at least 60%, or at least 65% of the population of expanded T cells expresses the chimeric receptor.

108. The method of any one of claims 1-107, wherein the population of expanded T cells comprises at least IxlO8, at least 2xl08, or at least 3xl08viable nucleated cells.

109. The method of any one of claims 1-108, wherein the chimeric receptor is a chimeric antigen receptor (CAR) comprising an extracellular binding domain comprising an antigen binding site that specifically binds a cell-surface protein, wherein the antigen-binding site comprises a heavy chain variable domain (VH) comprising complementarity determining regions CDRHI, CDRH2, and CDRH3 and a light chain variable domain (VL) comprising complementarity determining regions CDRLI, CDRL2, and CDRLI.

110. The method of claim 109, wherein the antigen-binding site specifically binds a B-cell surface protein.

111. The method of claim 110, wherein the B-cell surface protein is CD19.

112. The method of any one of claims 109-111, wherein the antigen-binding site is humanized or fully human.

113. The method of any one of claims 109-112, wherein:(i) the CDRHI , CDRH2, and CDRH3 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 CDRL3 comprise the amino acid sequences of SEQ ID NO: 5, YDD, and SEQ ID NO: 7, respectively; or(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 CDRL3 comprise the amino acid sequences of SEQ ID NO: 14, HTS, and SEQ ID NO: 16, respectively.

114. The method of any one of claims 109-113 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; or(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.

115. The method of any one of claims 109-114, wherein:(i) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 4 and 8, respectively; or(ii) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 13 and 17, respectively.

116. The method of any one of claims 109-115, wherein the antigen-binding site is present in an scFv.

117. The method of claim 116, 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 and 18.

118. The method of claim 116 or 117, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 9 and 18.1 19. The method of any one of claims 109- 1 18, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain.

120. The method of claim 119, wherein the transmembrane domain comprises a CD8 alpha chain transmembrane domain.

121. The method of claim 120, wherein the CD8 alpha chain transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19.

122. The method of any one of claims 119-121, wherein the costimulatory domain comprises a 4- IBB intracellular domain.

123. The method of claim 122, wherein the 4- IBB intracellular domain comprises the amino acid sequence of SEQ ID NO: 20.

124. The method of any one of claims 119-123, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

125. The method of claim 124, wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 21.

126. The method of any one of claims 119-125, wherein the CAR further comprises a hinge domain or linker interposed between the extracellular binding domain and the transmembrane domain.

127. The method of claim 126, wherein the hinge domain is a CD8 alpha chain hinge.

128. The method of claim 127, wherein the CD8 alpha chain hinge comprises the amino acid sequence of SEQ ID NO: 22.

129. The method of any one of claims 119-128, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 23 and 27.

130. The method of any one of claims 109-118, wherein the CAR further comprises a killer immunoglobulin-like receptor (KIR) transmembrane domain and a KIR cytoplasmic domain.

131. A composition of engineered T cells generated by the method of any one of claims 1- 130.

132. The composition of claim 131, further comprising a pharmaceutically acceptable carrier or excipient.

133. A method of treating a disease in a patient in need thereof, the method comprising administering to the patient the composition of claim 131 or 132.

134. The method of claim 133, wherein the engineered T cells are autologous to the patient.

135. The method of claim 133 or 134, wherein the method comprises administering the engineered T cells to the patient at a dose from 1 x 105cells / kg to 1 x 108cells / kg.

136. The method of claim 135, wherein the dose is from 1 x 106cells / kg to 1 x 107cells / kg.

137. The method of any one claims 133-136, wherein the disease is an autoimmune disease.

138. The method of claim 137, wherein the autoimmune disease is a B-cell-mediated autoimmune disease.

139. The method of claim 137 or 138, 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.

140. The method of any one of claims 137-139, 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, juvenile myositis, systemic sclerosis with skin involvement, systemic sclerosis with severe organ involvement, and immune mediated necrotizing myopathy.

141. The method of any one claims 133-136, wherein the disease is cancer.

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