Treatment of lupus nephritis using anti-CD19 CAR-T cell therapy

Abstract

Provided herein are methods and compositions for treating subjects with B-cell-associated autoimmune diseases, such as lupus nephritis, using T cells engineered with a chimeric antigen receptor that binds CD19.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 419,193, filed October 25, 2022, International (PCT) Application No. PCT / US2023 / 010034, filed January 3, 2023, U.S. Provisional Patent Application No. 63 / 512,061, filed July 5, 2023, U.S. Provisional Patent Application No. 63 / 518,745, filed August 10, 2023, and U.S. Provisional Patent Application No. 63 / 587,329, filed October 2, 2023, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] Technical Field The present invention relates generally to T cells engineered to express chimeric antigen receptors (CARs) (e.g., fully human anti-CD19 CARs) and their use in the treatment and / or prevention of autoimmune diseases such as lupus nephritis. [Background technology]

[0003] background Treatment for autoimmune diseases typically requires multiple treatments to control the disease. The most common treatments are corticosteroids and cytotoxic drugs, which can be highly toxic. These drugs suppress the entire immune system, potentially leading to serious infections and harmful side effects on the bone marrow, liver, and / or kidneys. Therefore, their long-term use and the use of multiple drugs are limited. Many patients develop severe, potentially life-threatening symptoms, requiring close monitoring and aggressive treatment.

[0004] Current treatment regimens for B-cell-mediated autoimmune diseases, such as lupus nephritis, include high-dose glucocorticoids in combination with immunosuppressants such as cyclophosphamide (CYC) and rituximab to induce remission and prevent further organ damage. However, relapses are common and remain a major challenge in clinical management. Disease management involves the use of low-dose glucocorticoids in combination with broad-spectrum immunosuppressants, such as azathioprine (AZA), methotrexate, and mycophenolate mofetil (MMF), to maintain disease control. Repeated administration of rituximab or CYC may be required upon recurrence of disease activity. Despite these treatments, however, there remains a significant unmet need for novel therapies that can easily achieve sustained remission, effectively resolve the frequent relapses associated with existing therapies, and reduce the extensive background immunosuppressive therapy required to maintain good disease control. Summary of the Invention

[0005] overview Provided herein are methods and compositions for treating a subject with an autoimmune disease or disorder. Of note, provided herein are methods and compositions for treating a subject with an autoimmune disease or disorder for which conventional treatment regimens have been shown to be ineffective (e.g., due to toxicity profiles or limited pharmacological activity). In some embodiments, the conventional treatment regimen (i.e., standard of care (SOC) therapy) is discontinued, such that the patient does not receive any supplemental immunomodulatory agents for a period of time (e.g., at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 1 year, 2 years, 3 years, or more) after administration of the provided methods and compositions. In some embodiments, the conventional treatment regimen (i.e., SOC therapy) is discontinued prior to harvesting the host cells (e.g., lymphocytes such as T cells) to be engineered by any of the methods described herein (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, or more prior to harvesting the host cells). Further provided herein are methods and compositions for treating a subject having an autoimmune disease or disorder, wherein the methods and compositions are used in place of known conventional treatment regimens. The present disclosure further provides methods and compositions for treating a subject having a B cell-associated autoimmune disease or disorder. In some embodiments, the B cell-associated autoimmune disease or disorder comprises systemic lupus erythematosus. In some embodiments, the B cell-associated autoimmune disease is lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is class III or class IV lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is Class III lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is Class IV lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is Class II lupus nephritis. In some embodiments, the present disclosure provides methods and compositions that result in the removal of B cells from tissue sites that are not normally accessible to conventional therapies.

[0006] The methods of the present disclosure use T cells engineered to express an anti-CD19 CAR construct, which can reduce or deplete B cells responsible for an autoimmune disease or disorder in a patient. In many embodiments, the anti-CD19 CAR construct has a reduced toxicity profile compared to conventional therapies. In some embodiments, the anti-CD19 CAR is substantially non-toxic to the subject undergoing CAR therapy treatment. In some embodiments, such low- or non-toxic CAR therapies provided by the present disclosure allow for higher and / or multiple doses, resulting in B cell depletion in areas that cannot be treated with conventional autoimmune therapies due to their toxicity profiles. Indeed, the CAR therapies provided herein demonstrate significantly reduced toxicity commonly associated with CAR therapy (e.g., anti-CD19 CAR therapy), including cytokine release syndrome (CRS) and neurotoxicity. In some embodiments, the present disclosure provides methods and compositions for treating a B cell-related autoimmune disease or disorder (e.g., lupus nephritis, such as class II, class III, or class IV lupus nephritis) using an anti-CD19 CAR. In many embodiments of any of the methods and compositions provided herein, the anti-CD19 CAR comprises a fully human chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the intracellular domain comprising a cytoplasmic signaling domain and one or more costimulatory domains. See U.S. Patent No. 10,287,350, incorporated herein by reference in its entirety.

[0007] The present disclosure provides, inter alia, methods for treating lupus nephritis (e.g., class II, class III, or class IV lupus nephritis), comprising administering to a subject in need of treatment a therapeutically effective amount of T cells comprising a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises, from N-terminus to C-terminus, (a) an antigen-binding fragment of an anti-CD19 antibody, (b) a transmembrane domain, and (c) an intracellular T cell signaling domain derived from human CD3ζ.

[0008] In some embodiments, the anti-CD19 antibody is a human antibody.

[0009] In some embodiments, the antigen-binding fragment of an anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0010] In some embodiments, the antigen-binding fragment of an anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 3, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0011] In some embodiments, the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 8.

[0012] In some embodiments, the antigen-binding fragment of the anti-CD19 antibody comprises the amino acid sequence of SEQ ID NO:17.

[0013] In some embodiments, the transmembrane domain is derived from human CD8.

[0014] In some embodiments, the transmembrane domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:11.

[0015] In some embodiments, the intracellular T cell signaling domain derived from human CD3ζ comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 23. In some embodiments, the intracellular T cell signaling domain derived from human CD3ζ comprises the amino acid sequence of SEQ ID NO: 23.

[0016] In some embodiments, the CAR further comprises an intracellular T cell signaling domain derived from human CD28. In some embodiments, the intracellular T cell signaling domain derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 21.

[0017] In some embodiments, the CAR does not comprise an intracellular T cell signaling domain derived from 4-1BB.

[0018] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 10 or 13.

[0019] In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector further comprises a U3 promoter of murine stem cell virus (MSCV) operably linked to the nucleic acid.

[0020] In some embodiments, at least 10% (e.g., 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%, or at least 99%) of the provided T cells express a CAR (e.g., any CAR provided herein). In some embodiments, the provided T cells comprise at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%) CD8+ cytotoxic T cells. In some embodiments, the provided T cells comprise at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%) CD4+ helper T cells.

[0021] In some embodiments, the lupus nephritis is Class III or Class IV active, biopsy-proven, proliferative lupus nephritis according to the 2018 ISN / RPS criteria. In some embodiments, the lupus nephritis is Class III active, biopsy-proven, proliferative lupus nephritis according to the 2018 ISN / RPS criteria. In some embodiments, the lupus nephritis is Class IV active, biopsy-proven, proliferative lupus nephritis according to the 2018 ISN / RPS criteria. In some embodiments, the lupus nephritis is Class II active, biopsy-proven, proliferative lupus nephritis according to the 2018 ISN / RPS criteria.

[0022] In some embodiments, the subject tests positive for antinuclear antibodies, e.g., has an antinuclear antibody titer of 1:80 or greater as measured by Hep-2 immunofluorescence assay or enzyme immunoassay. In some embodiments, the subject tests positive for anti-dsDNA antibodies, e.g., has an anti-dsDNA antibody concentration of 30 IU / ml or greater as measured by enzyme-linked immunosorbent assay. In some embodiments, the subject tests positive for anti-Smith antibodies. In some embodiments, the subject tests positive for antiphospholipid antibodies. In some embodiments, the subject tests positive for antiphospholipid antibodies and exhibits clinical symptoms consistent with antiphospholipid syndrome.

[0023] In some embodiments, the therapeutically effective amount is about 5×10 7 ~1×10 8 pieces, about 5×10 7 ~9×10 7 pieces, about 5×10 7 ~8×10 7 pieces, about 5×10 7 ~7×10 7 pieces, about 5×10 7 ~6×10 7 pieces, about 6×10 7 ~1×10 8 pieces, about 7×10 7 ~1×10 8 pieces, about 8×10 7 ~1×108 pieces, approximately 9×10 7 ~1×10 8 pieces, about 6×10 7 ~9×10 7 pieces, or approximately 7 x 10 7 ~8×10 7 In some embodiments, the therapeutically effective amount is in the range of about 5 x 10 T cells. 7 ~1×10 8 In some embodiments, the therapeutically effective amount is in the range of about 5 x 10 T cells. 7 T cells (e.g., 5 x 10 7 In some embodiments, the therapeutically effective amount is about 1 x 10 8 T cells (e.g., 1 x 10 8 T cells).

[0024] In some embodiments, the provided T cells are administered by intravenous infusion.

[0025] In some embodiments, the subject is administered a single dose of T cells.

[0026] In some embodiments, the subject has undergone lymphocyte depletion therapy. In some embodiments, lymphocyte depletion therapy includes administration of cyclophosphamide (e.g., at a dose of 300 mg / m), e.g., once daily for 3 days prior to administration of the T cells, starting 5-7 days prior to administration of the T cells. 2 ) and fludarabine (e.g., at a dose of 30 mg / m 2 ) intravenously. In some embodiments, the subject does not receive lymphocyte depletion therapy prior to administration of the T cells. In some embodiments, the subject has received minimal lymphocyte depletion therapy. In some embodiments, the initial lymphocyte depletion therapy includes administering cyclophosphamide (e.g., at a dose of 150 mg / m) to the subject, e.g., once daily for three days prior to administration of the T cells, starting 5-7 days prior to administration of the T cells. 2 ) and fludarabine (e.g., at a dose of 15 mg / m 2In some embodiments, the minimal lymphocyte-depleting therapy results in about a 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% reduction in lymphocytes in the subject compared to the subject prior to administration of the lymphocyte-depleting therapy or another suitable control. In some embodiments, the subject has undergone minimal lymphocyte-depleting therapy, such that the subject has about a 50% reduction in lymphocytes compared to the subject prior to administration of the lymphocyte-depleting therapy or another suitable control.

[0027] All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or information statement was specifically and individually indicated to be incorporated by reference. In the event that the statements in the publications, patents, patent applications, and information incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting statements.

[0028] When values ​​are described as ranges, it is understood that the description includes disclosure of all possible subranges within that range, as well as specific numerical values ​​falling within that range, whether or not a specific numerical value or specific subrange is explicitly recited.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in accordance with the present disclosure, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, and in the event of a conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0030] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0031] [Figure 1] 1 is a series of graphs showing the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) from a systemic lupus erythematosus (SLE) patient transduced with Hu19-CD828Z chimeric antigen receptor (CAR) construct against NALM6 (CD19+) cells. [Figure 2] 1 is a series of graphs showing the cytotoxic activity of PBMCs from SLE patients and healthy donors transduced with the Hu19-CD828Z CAR construct against autologous primary B cells expressing CD19. [Figure 3A] This is a graph showing the release of interferon gamma (IFNγ) after co-culture of PBMCs from SLE patients transduced with the Hu19-CD828Z CAR construct with NALM6 (CD19+) tumor cells. [Figure 3B] This is a graph showing the release of interferon gamma (IFNγ) after PBMCs from SLE patients transduced with the Hu19-CD828Z CAR construct were co-cultured with autologous primary B cells expressing CD19. [Figure 4A] Graph showing proliferation of Hu19-CD828Z transduced PBMCs after co-culture with NALM6 (CD19+) tumor cells. [Figure 4B] 1 is a graph showing proliferation of Hu19-CD828Z transduced PBMCs after co-culture with autologous primary B cells expressing CD19. [Figure 4C] Graph showing proliferation of Hu19-CD828Z transduced PBMCs after co-culture with control K562 (CD19-) cells. [Figure 5] FIG. 1 is a schematic diagram of a lentiviral vector encoding an anti-CD19 CAR transgene. DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed Description definition As used herein, the term "about" a number refers to an inclusive range of 10% below that number to 10% above that number. "About" a range refers to 10% below the lower limit of the range to 10% above the upper limit of the range.

[0033] As used herein, the term "antibody" refers to any immunoglobulin, whether naturally occurring or wholly or partially synthetically produced. The term also includes all derivatives thereof that maintain specific binding ability. In some embodiments, the term "antibody" refers to any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. Antibody proteins may be derived from natural sources or may be partially or fully synthetic. Antibodies are classified as monoclonal and polyclonal. Antibodies may belong to any immunoglobulin class, including the human immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. In certain embodiments, an antibody may be a member of the IgG immunoglobulin class.

[0034] As used herein, "derived from" or "derivative" refers to structural and functional similarity (e.g., between polynucleotides, polypeptides) between a molecule of interest and a reference molecule. With respect to structural similarity, the molecule of interest does not necessarily contain the same sequence (e.g., nucleic acid sequence, amino acid sequence, etc.) as the reference molecule, but has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence (e.g., nucleic acid sequence, amino acid sequence, etc.) of the reference molecule or a fragment thereof comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence of the reference molecule. With respect to functional similarity, a subject molecule has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of a reference molecule or fragment thereof as determined by a suitable assay. For example, a subject polypeptide is considered to be derived from a reference polypeptide if it is structurally similar to the reference polypeptide, as defined above, and retains a particular function(s), such as a particular inter- or intramolecular interaction (e.g., binding to a protein such as a particular receptor, or signaling activity), although such interaction may be stronger, similar, or weaker than that of the reference polypeptide. As another non-limiting example, a subject polynucleotide can be considered to be derived from a reference polynucleotide if the subject polynucleotide is structurally similar to the reference polynucleotide, as defined above, encodes a protein or protein fragment that is a derivative of the protein encoded by the reference polynucleotide, or has the same or similar function as the reference polynucleotide (e.g., a regulatory element, e.g., a promoter or enhancer). Functional similarity is considered in the context of the present disclosure.For example, when applied to a subject intracellular T cell signaling domain derived from a reference protein (e.g., CD3ζ, CD28), the subject intracellular T cell signaling domain is structurally and functionally similar to the intracellular T cell signaling domain of the reference protein known in the art. Similarly, when applied to a subject transmembrane domain derived from a reference protein, the subject transmembrane domain is structurally and functionally similar to the transmembrane domain of the reference protein known in the art. In one non-limiting example, the intracellular T cell signaling domain derived from a CD3ζ molecule retains sufficient CD3ζ structure such that it is capable of transducing a signal (e.g., ZAP-70 activation) under appropriate conditions.

[0035] As used herein, the term "functional fragment" of a reference biomolecule (e.g., a polynucleotide or polypeptide) refers to a shorter and / or smaller derivative of the reference biomolecule that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of the fragment of the reference biomolecule.

[0036] As used herein, the term "operably linked" refers to polynucleotide sequences placed in a functional relationship with each other. For example, a promoter or enhancer is operably linked to a coding sequence if it regulates or contributes to the regulation or transcription of the coding sequence. Operatively linked DNA sequences encoding regulatory sequences are usually contiguous to the coding sequence. However, enhancers can function up to several kilobases or more away from the promoter. Furthermore, multicistronic constructs can contain multiple coding sequences using only one promoter by including 2A self-cleaving peptides, IRES elements, etc. Thus, some polynucleotide elements may be operably linked but not contiguous.

[0037] As used herein, the terms "patient" or "subject" are used interchangeably to refer to any organism to which the compositions disclosed herein can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient or subject is a human. In many embodiments, the patient is a human subject with an autoimmune disease or disorder.

[0038] Methods and compositions for treating autoimmune diseases The present disclosure provides methods and compositions that can be used to treat a subject identified as having an autoimmune disease. The present disclosure further provides methods and compositions for treating a B cell-associated autoimmune disease. In some embodiments, the B cell-associated autoimmune disease is systemic lupus erythematosus. In some embodiments, the B cell-associated autoimmune disease is lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is class III or class IV lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is class III lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is class IV lupus nephritis. In some embodiments, the B cell-associated autoimmune disease is class II lupus nephritis.

[0039] The present disclosure provides, inter alia, methods and compositions for reducing the number of B cells in the tissues of a subject with an autoimmune disease. The present disclosure also provides methods and compositions for treating a subject with an autoimmune disease. Additionally, the present disclosure provides engineered T cells (e.g., T cells engineered to express any of the CARs described herein) and methods of making engineered T cells. The present disclosure also provides engineered nucleic acids (e.g., engineered nucleic acid constructs) encoding engineered polypeptides (e.g., any of the engineered polypeptides, e.g., CARs, described herein).

[0040] The present disclosure recognizes that B cells express a variety of cell surface molecules, such as CD19, during differentiation and proliferation. CD19 is widely expressed on B cells at all stages of B cell development, from pro-B cells to plasmablasts. The present disclosure further recognizes that because CD19 is ubiquitous on B cells, CD19 can serve as a therapeutic target for certain methods and compositions (e.g., methods and compositions for treating autoimmune diseases). Accordingly, in some embodiments, provided herein are methods and compositions for reducing the number of B cells in a subject (e.g., in a tissue of a subject) by targeting CD19. In some embodiments, the present disclosure provides methods and compositions for treating a subject with a B cell-associated autoimmune disease by targeting CD19. In some embodiments, the present disclosure provides engineered T cells that target CD19. In some embodiments, the present disclosure provides engineered nucleic acids that express one or more polypeptides that target CD19. In many embodiments of the present disclosure, a CAR that binds to CD19 is used to target cells (e.g., B cells) that express CD19.

[0041] Chimeric Antigen Receptor In some embodiments, a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain is or comprises an antigen-binding domain (e.g., a CD19-binding domain such as an anti-CD19 scFv). In some embodiments, the transmembrane domain is or comprises a transmembrane domain or functional fragment thereof derived from any suitable cell membrane-associated polypeptide, e.g., derived from a membrane-bound or transmembrane polypeptide. In some embodiments, the transmembrane domain is or comprises a transmembrane domain or functional fragment thereof derived from a T cell receptor α chain, a T cell receptor β chain, a CD3 ζ chain, a CD28 polypeptide, or a CD8 polypeptide (e.g., a CD8α polypeptide). In some embodiments, the intracellular domain is or comprises an intracellular signaling domain (e.g., any of the intracellular signaling domains described herein, e.g., derived from a CD28 or CD3 polypeptide). In some embodiments, the intracellular signaling domain comprises one or more signaling sequences or motifs. In some embodiments, the one or more signaling sequences or motifs are essential for the functional signaling capability of the polypeptide (e.g., the intracellular signaling domain). In some embodiments, the signaling sequence is a sequence derived from a CD3 polypeptide (e.g., a CD3ζ polypeptide). In some embodiments, the signaling sequence is derived from a CD28 polypeptide. In some embodiments, the signaling sequence is or includes a costimulatory domain (e.g., any costimulatory domain described herein, e.g., derived from a CD28 polypeptide). In some embodiments, the CAR of the present disclosure is a human CAR.

[0042] Extracellular domain In some embodiments, the extracellular domain used in accordance with the present disclosure comprises an antigen-binding domain (e.g., any antigen-binding domain described herein). In some embodiments, the antigen-binding domain is or comprises an antibody sequence (e.g., an immunoglobulin) or antigen-binding fragment thereof (e.g., any antibody or antigen-binding fragment thereof described herein). Anticalins or other alternative scaffolds are also contemplated.

[0043] In some embodiments, the antigen-binding domain is one or more of a Fab, a Fab', a F(ab'), an Fv, a domain antibody (dAb), a single-chain antibody (scFv), a chimeric antibody, a diabody, a triabody, a tetrabody, a scAb, or a single-domain antibody (e.g., a V H H or V NAR ) polypeptide sequence. In some embodiments, the antigen-binding domain comprises at least a portion of an immunoglobulin (e.g., an antibody fragment comprising an antigen-binding portion) sufficient to confer specific antigen-binding properties to the polypeptide. In some embodiments, the antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises the VH and VL domains of an antibody. In some embodiments, the scFv comprises a spacer sequence between the VH and VL. In some embodiments, the scFv comprises the spacer sequence set forth in SEQ ID NO: 9 between the VH and VL. In some embodiments, the antigen-binding domain is humanized or fully human (e.g., derived from an appropriate human polypeptide). Exemplary methods for generating fully human antibodies are described in Lu et al., (2020) J. Biomed. Sci. (2020) 27(1):1.

[0044] In some embodiments, the antigen-binding domain binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain specifically binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain binds to a CD19 polypeptide (e.g., a CD19 polypeptide present on the surface of a cell, such as a B cell). In some embodiments, the antigen-binding domain specifically binds to a CD19 polypeptide. In some embodiments, the antigen-binding domain comprises an antibody, or antigen-binding fragment thereof, that binds to a CD19 polypeptide. In some embodiments, the antigen-binding domain comprises an scFv sequence (e.g., an anti-CD19 scFv) that binds to a CD19 polypeptide.

[0045] It is generally understood that CD19 expression is primarily restricted to B lymphocytes. CD19 has two N-terminal extracellular Ig-like domains separated by a non-Ig-like domain, a hydrophobic transmembrane domain, and a large C-terminal cytoplasmic domain. The CD19 protein forms a complex with several membrane proteins, such as complement receptor type 2 (CD21) and tetraspanin (CD81), which lowers the threshold for antigen-initiated B cell activation. Activation of this B cell antigen receptor complex activates the phosphatidylinositol 3-kinase signaling pathway, which subsequently releases calcium ions from intracellular stores. Examples of human CD19 polypeptide sequences include, but are not limited to, NCBI Reference Sequence: NP_001171569.1 and its fragments and derivatives.

[0046] In some embodiments, the antigen-binding domain comprises the variable region of an anti-CD19 antibody. In some embodiments, the antigen-binding domain comprises the variable region of an anti-CD19 monoclonal antibody. In some embodiments, the antigen-binding domain comprises the variable region of a murine or human anti-CD19 monoclonal antibody. The anti-CD19 monoclonal antibody can be obtained or derived from a subject (e.g., a mouse, a rat, a rabbit, a human, etc.) using any suitable method. In some aspects, the antigen-binding domain comprises the light chain variable region and the heavy chain variable region of a murine, human, or humanized anti-CD19 monoclonal antibody. In some embodiments, the antigen-binding domain comprises the light chain variable region of a murine, human, or humanized anti-CD19 monoclonal antibody. In some embodiments, the antigen-binding domain comprises the heavy chain variable region of a murine, human, or humanized anti-CD19 monoclonal antibody. The 47G4 antibody (described in US Patent Application Publication No. 2010 / 0104509, which is incorporated herein by reference in its entirety) is an example of a human anti-CD19 monoclonal antibody that can be used in accordance with the present disclosure.

[0047] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0048] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:3, respectively, and the light chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:3, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0049] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0050] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:3, respectively. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:3, respectively.

[0051] In some embodiments, the antigen-binding domain that binds to CD19 comprises a light chain variable domain, wherein the light chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the antigen-binding domain that binds to CD19 comprises a light chain variable domain, wherein the light chain variable domain comprises CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0052] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 7, and the light chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least at least 95% sequence identity to SEQ ID NO: 7, and the light chain variable domain comprises an amino acid sequence having at least at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7, and the light chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0053] In some embodiments, the antigen binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 7. In some embodiments, the antigen binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen binding domain that binds to CD19 comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0054] In some embodiments, the antigen binding domain that binds to CD19 comprises a light chain variable domain, wherein the light chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 8. In some embodiments, the antigen binding domain that binds to CD19 comprises a light chain variable domain, wherein the light chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen binding domain that binds to CD19 comprises a light chain variable domain, wherein the light chain variable domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen binding domain that binds to CD19 comprises a light chain variable domain, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0055] In some embodiments, the antigen-binding domain that binds to CD19 comprises a spacer sequence between the two domains or components. In some aspects, the antigen-binding domain comprises a spacer sequence between the heavy chain variable region and the light chain variable region. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO:9.

[0056] In some embodiments, the antigen-binding domain that binds to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 17. In some embodiments, the antigen-binding domain that binds to CD19 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, the antigen-binding domain that binds to CD19 comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0057] In some embodiments, the antigen-binding domain that binds to CD19 is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 18. In some embodiments, the antigen-binding domain that binds to CD19 is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, the antigen-binding domain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 18.

[0058] Other antigen-binding domains that bind to CD19 can also be included in the CARs disclosed herein. Exemplary antigen-binding domains are described in International Application Publication No. WO2017062952 and U.S. Application Publication No. US20220220200.

[0059] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 35, 36, and 37, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 38, 39, and 40, respectively. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 29, and the light chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 30.

[0060] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 41, 42, and 43, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 44, 45, and 46, respectively. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 31, and the light chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 32.

[0061] In some embodiments, the antigen-binding domain that binds to CD19 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 47, 48, and 49, respectively, and the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 50, 51, and 52, respectively. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 33, and the light chain variable domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 34.

[0062] In some embodiments, the extracellular domain of the CAR further comprises a hinge region. In some embodiments, the hinge region is located between (e.g., connects) the extracellular domain and the transmembrane domain. In some embodiments, the hinge region is a short sequence of amino acids that can increase structural flexibility between polypeptide domains, for example, between the extracellular domain and the transmembrane domain (see, e.g., Woof et al., Nat. Rev. Immunol. 4(2):89-99 (2004)). In some embodiments, the hinge region can comprise all or a portion of the extracellular region of any suitable transmembrane protein (e.g., CD8α).

[0063] In some embodiments, the hinge region is derived from a CD8α protein or a CD28 protein. In some embodiments, the hinge region is derived from a CD8α protein. In some embodiments, the hinge domain is derived from a CD28 protein. In some embodiments, the hinge region is or comprises a hinge region from a CD28 protein, or a functional fragment thereof. In some embodiments, the hinge region is or comprises a hinge region from a CD8α protein, or a functional fragment thereof. In some embodiments, the hinge region is derived from a human CD8α protein or a human CD28 protein. In some embodiments, the hinge region is derived from a human CD8α protein. In some embodiments, the hinge region is or comprises a hinge region from a human CD28 protein, or a functional fragment thereof. In some embodiments, the hinge region is or comprises a hinge region from a human CD8α protein, or a functional fragment thereof.

[0064] In some embodiments, the hinge region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 28. In some embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0065] In some embodiments, the hinge region is derived from the same polypeptide as the transmembrane domain. In some embodiments, the hinge region and transmembrane domain are derived from a CD8 polypeptide. In some embodiments, the hinge region and transmembrane domain are derived from a CD8α polypeptide. In some embodiments, the hinge region and transmembrane domain comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 19. In some embodiments, the hinge region and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, the hinge region and transmembrane domain comprise the amino acid sequence set forth in SEQ ID NO: 19.

[0066] In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 20. In some embodiments, the hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, the hinge region and transmembrane domain are encoded by the nucleic acid sequence set forth in SEQ ID NO: 20.

[0067] Transmembrane domain In some embodiments, the transmembrane domain of the CAR is derived from a natural source (e.g., a naturally occurring or wild-type polypeptide). In some embodiments, the transmembrane domain used in accordance with the present disclosure is derived from any suitable transmembrane protein or polypeptide known in the art. In some embodiments, the transmembrane domain is derived from a CD3ε polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45 polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD134 polypeptide, a CD137 polypeptide, a CD154 polypeptide, a T cell receptor α chain polypeptide, a T cell receptor β chain polypeptide, a T cell receptor ζ chain polypeptide, or any combination thereof. In some embodiments, the transmembrane is or comprises a transmembrane domain or functional fragment thereof derived from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45 polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD134 polypeptide, a CD137 polypeptide, a CD154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any derivative thereof and / or any combination thereof. In some embodiments, the transmembrane structure is synthetically derived or engineered. In some embodiments, synthetically derived or engineered transmembrane domains comprise primarily hydrophobic residues (e.g., leucine, valine, etc.). In some embodiments, the engineered transmembrane domain is or comprises any engineered transmembrane domain known in the art.

[0068] The present invention recognizes that CD8 is a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR) and is expressed exclusively on the surface of T cells (e.g., cytotoxic T cells). The most common form of CD8 exists as a dimer consisting of the CD8 α chain and the CD8 β chain. In some embodiments, the transmembrane domain is derived from the CD8 α protein. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO:11. In some embodiments, the transmembrane protein comprises the amino acid sequence set forth in SEQ ID NO:11.

[0069] The present invention further recognizes that CD28 is expressed on T cells and provides a costimulatory signal necessary for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2). In some embodiments, a CAR of the present disclosure comprises a CD28 transmembrane domain. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane protein comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0070] Intracellular signaling domains In some embodiments, the intracellular signaling domain of a CAR disclosed herein is derived from a polypeptide found in a human (e.g., an intracellular signaling domain found in any suitable human polypeptide, or a fragment thereof). In some embodiments, the intracellular signaling domain provided herein is derived from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3γ polypeptide, a CD3δ polypeptide, a CD3ζ polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FcεRI polypeptide (e.g., an FcεRI gamma chain polypeptide), an FcγRI polypeptide, a LIGHT polypeptide, an NKG2C polypeptide, an OX40 polypeptide, a PD-1 polypeptide, or any derivative thereof or any combination thereof. In some embodiments, the intracellular signaling domain is derived from a CD3ζ polypeptide. In some embodiments, the intracellular signaling domain is derived from a CD28 polypeptide. In some embodiments, the intracellular signaling domain is derived from a CD28 polypeptide and a CD3ζ polypeptide.

[0071] In some embodiments, the intracellular signaling domain comprises at least one intracellular signaling domain or functional fragment thereof derived from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3γ polypeptide, a CD3δ polypeptide, a CD3ζ polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FcεRI polypeptide (e.g., an FcεRI gamma chain polypeptide), an FcγRI polypeptide, a LIGHT polypeptide, an NKG2C polypeptide, an OX40 polypeptide, a PD-1 polypeptide, or any derivative or any combination thereof. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof derived from a CD3ζ polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof derived from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof derived from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof derived from a CD3ζ polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, an intracellular signaling domain derived from a CD28 polypeptide, or a functional fragment thereof, and an intracellular signaling domain derived from a CD3ζ polypeptide, or a functional fragment thereof.

[0072] In some embodiments, an intracellular signaling domain of the present disclosure comprises at least one signaling sequence derived from at least one intracellular signaling domain or functional fragment thereof from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3γ polypeptide, a CD3δ polypeptide, a CD3ζ polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FcεRI polypeptide (e.g., an FcεRI gamma chain polypeptide), an FcγRI polypeptide, a LIGHT polypeptide, an NKG2C polypeptide, an OX40 polypeptide, a PD-1 polypeptide, or any combination thereof. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from a CD3ζ polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence derived from a CD28 polypeptide and at least one signaling sequence derived from a CD3ζ polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, at least one signaling sequence derived from a CD28 polypeptide and at least one signaling sequence derived from a CD3ζ polypeptide.

[0073] In some embodiments, the intracellular signaling domain comprises at least one signaling sequence or signaling motif. In some embodiments, the signaling sequence (or signaling motif) comprises one or more (e.g., two, three, four, five, or more) costimulatory domains (e.g., any costimulatory domain described herein). In some embodiments, the signaling sequence comprises one costimulatory signaling domain. In some embodiments, the signaling sequence comprises two costimulatory signaling domains. In some embodiments, the signaling sequence comprises three costimulatory signaling domains. In some embodiments, the signaling sequence comprises two or more of the same costimulatory signaling domains. In some embodiments, the signaling sequence comprises two or more different costimulatory signaling domains.

[0074] In some embodiments, a signaling sequence used in accordance with the present disclosure is or includes one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the signal sequence is or includes the consensus sequence YXXL / I, where Y is a tyrosine residue, L / I is a leucine or isoleucine residue, and X is any amino acid residue. In some embodiments, the signal sequence is YXXL / IX (6~8) YXXL consensus sequence, where Y is a tyrosine residue, L / I is a leucine or isoleucine residue, and X is any amino acid residue. In some embodiments, the signaling sequence comprises a YNMN motif. In some embodiments, the signaling sequence comprises at least one ITAM sequence derived from a CD3 polypeptide (e.g., a CD3ζ polypeptide). In some embodiments, the signaling sequence comprises at least one ITAM sequence derived from a CD28 polypeptide.

[0075] The most common intracellular signaling domain used in CAR therapy is understood to be the intracellular signaling domain of CD3 zeta (CD3ζ). CD3 zeta binds to a T cell receptor to generate a signal and contains ITAM. In some embodiments, the intracellular signaling domain is or comprises a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a CD3ζ polypeptide or a functional fragment thereof.

[0076] In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 23. In some embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0077] In some embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 24. In some embodiments, the intracellular signaling domain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 24.

[0078] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD28. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain from a CD28 polypeptide or a functional fragment thereof. In some embodiments, the intracellular signaling domain of a CD28 polypeptide or a functional fragment thereof comprises a costimulatory signaling domain.

[0079] In some embodiments, the intracellular signaling domain disclosed herein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0080] In some embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 22. In some embodiments, the intracellular signaling domain is encoded by the nucleic acid sequence set forth in SEQ ID NO: 22.

[0081] Chimeric antigen receptor (CAR) In some embodiments, a CAR of the present disclosure comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises a signal peptide sequence (also referred to as a targeting signal, localization signal, localization sequence, leader sequence, or leader peptide), an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises, from N-terminus to C-terminus, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises, from N-terminus to C-terminus, a signal peptide sequence, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the signal peptide sequence is cleaved from the CAR during or after insertion into a membrane (e.g., the ER membrane) during synthesis of the CAR protein. In some embodiments, the domains or components of the CAR (e.g., the extracellular domain, hinge region, transmembrane domain, intracellular signaling domain, etc.) are directly linked or adjacent to each other. In some embodiments, the domains or components of the CAR are not directly linked or adjacent to each other.

[0082] In some embodiments, a CAR described herein comprises an intracellular signaling domain, wherein the intracellular signaling domain comprises (a) an intracellular signaling domain of CD3ζ, or a functional fragment thereof, and (b) at least one of an intracellular signaling domain of 4-1BB, OX40, or CD28, or a functional fragment thereof. In some embodiments, the intracellular signaling domain of 4-1BB, or a functional fragment thereof, the intracellular signaling domain of OX40, and / or the intracellular signaling domain of CD28, or a functional fragment thereof, is or comprises a costimulatory domain.

[0083] In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD28, and (b) (i) an intracellular signaling domain comprising (a) the intracellular signaling domain of CD3ζ, or a functional fragment thereof, and (ii) the intracellular signaling domain of CD28, or a functional fragment thereof. In some embodiments, the intracellular signaling domain of CD28, or a functional fragment thereof, is or comprises a costimulatory signaling domain of CD28.

[0084] In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD8α, and (b) an intracellular signaling domain comprising (i) an intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) an intracellular signaling domain of CD28, FcεRIγ chain, and / or 4-1BB or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD8α, and (b) an intracellular signaling domain comprising (i) an intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) an intracellular signaling domain of CD28, FcεRIγ chain, and 4-1BB or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD8α, and (b) an intracellular signaling domain comprising (i) an intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) an intracellular signaling domain of FcεRIγ chain or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD8α, and (b) an intracellular signaling domain comprising (i) an intracellular signaling domain of CD3ζ, or a functional fragment thereof, and (ii) an intracellular signaling domain of 4-1BB, or a functional fragment thereof. In some embodiments, the intracellular signaling domain of CD28, or a functional fragment thereof, is or comprises a costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain of FcεRI, or a functional fragment thereof, is or comprises a costimulatory signaling domain of FcεRI. In some embodiments, the intracellular signaling domain of 4-1BB, or a functional fragment thereof, is or comprises a costimulatory signaling domain of 4-1BB.

[0085] In some embodiments, a CAR herein comprises (a) a transmembrane domain of CD8α, and (b) an intracellular signaling domain comprising (i)(a) an intracellular signaling domain of CD3ζ, or a functional fragment thereof, and (ii) an intracellular signaling domain of CD27 and / or CD28, or a functional fragment thereof. In some embodiments, the intracellular signaling domain of CD27, or a functional fragment thereof, is or comprises a costimulatory signaling domain of CD27. In some embodiments, the intracellular signaling domain of CD28, or a functional fragment thereof, is or comprises a costimulatory signaling domain of CD28.

[0086] In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD28, and (b)(i)(a) the intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) an intracellular signaling domain of CD27, 4-1BB, and / or FcεRIγ-chain or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD28, and (b)(i)(a) the intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) an intracellular signaling domain of CD27, 4-1BB, and FcεRIγ-chain or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD28, and (b)(i)(a) the intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) an intracellular signaling domain of CD27 or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD28 and (b) an intracellular signaling domain comprising (i)(a) the intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) the intracellular signaling domain of 4-1BB or a functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a transmembrane domain of CD28 and (b)(i)(a) the intracellular signaling domain of CD3ζ or a functional fragment thereof, and (ii) the intracellular signaling domain of the FcεRI γ-chain or a functional fragment thereof. In some embodiments, the intracellular signaling domain of CD27 or a functional fragment thereof is or comprises the costimulatory signaling domain of CD27. In some embodiments, the intracellular signaling domain of FcεRI or a functional fragment thereof is or comprises the costimulatory signaling domain of FcεRI. In some embodiments, the intracellular signaling domain of 4-1BB or a functional fragment thereof is or comprises the costimulatory signaling domain of 4-1BB.

[0087] The present disclosure also includes functional variants of any of the CARs or CAR domains / components described herein. Functional variants of CARs include, for example, variants of the CARs (parent CARs) described herein that retain the ability to recognize specific target cells to a similar extent, the same extent, or greater extent than the parent CAR. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional variant of the CAR can be, for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent CAR. In some embodiments, the parent CAR comprises the amino acid sequence set forth in SEQ ID NO: 10 or 13. Alternatively or additionally, in some embodiments, the functional variant of a CAR comprises the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In some embodiments, the non-conservative amino acid substitution does not reduce or inhibit the biological activity of the functional variant of the CAR. In some embodiments, the non-conservative amino acid substitution increases the biological activity of a functional variant of a CAR, thereby increasing the biological activity of the functional variant compared to its parent CAR.

[0088] The present invention further provides CARs that include an extracellular domain (e.g., including any of the known antigen binding domains, e.g., antibodies, scFvs, etc.) that targets any target molecule of interest, and further include any of the transmembrane domains described herein (including any of the hinge domains described herein) and any of the intracellular signaling domains described herein (including any of the signal sequences or motifs, any costimulatory domains, etc. described herein), present in any combination.

[0089] In some embodiments, the CAR comprises (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, and (c) an intracellular signaling domain comprising (i) the intracellular signaling domain of human CD3ζ or a fragment thereof, and (ii) the intracellular signaling domain of human CD28 or a fragment thereof, wherein the intracellular signaling domain of CD28 or a fragment thereof is or comprises a costimulatory domain. In some embodiments, the CAR comprises (a) a hinge region derived from a human CD8α polypeptide, (b) a transmembrane domain derived from a human CD8α polypeptide, and (c) an intracellular signaling domain comprising (i) the intracellular signaling domain of human CD3ζ, and (ii) the intracellular signaling domain of human CD28. In some embodiments, the CAR comprises the sequence set forth in SEQ ID NO: 27.

[0090] In some embodiments, the CAR comprises (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, and (c) an intracellular signaling domain comprising (i) the intracellular signaling domain of human CD3ζ or a fragment thereof, and (ii) the intracellular signaling domain of CD27 and / or CD28 or a fragment thereof, wherein the intracellular signaling domain of CD27 and / or CD28 or a fragment thereof is or comprises a costimulatory domain.

[0091] In some embodiments, the CAR comprises (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, and (c) an intracellular signaling domain comprising (i) the intracellular signaling domain of human CD3ζ or a fragment thereof, and (ii) the intracellular signaling domain of human CD28, human CD27, and / or the intracellular signaling domain of FcεRIγ chain or a fragment thereof, wherein the intracellular signaling domain of CD28, CD27, and / or the intracellular signaling domain of FcεRIγ chain or a fragment thereof is or comprises a costimulatory domain.

[0092] In some embodiments, the CAR comprises (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, and (c) an intracellular signaling domain comprising (i) the intracellular signaling domain of human CD3ζ, and (ii) the intracellular signaling domain of human CD28 and / or FcεRIγ chain, wherein the intracellular signaling domain of CD28 and / or FcεRIγ chain or a fragment thereof can be or comprise a costimulatory domain.

[0093] In some embodiments, the CAR described herein further comprises a signal peptide sequence. In some embodiments, the signal peptide is positioned at the amino terminus of the extracellular domain (e.g., the N-terminus of the antigen-binding domain). The signal peptide used in accordance with the present disclosure may comprise any suitable signal peptide sequence. In some embodiments, the signal peptide sequence is the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal peptide sequence or the signal peptide sequence of CD8α. In some embodiments, the CAR provided herein comprises a human scFv comprising the signal peptide sequence of CD8α. In some embodiments, the signal peptide sequence comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0094] In some embodiments, a provided CAR comprises (a) a hinge region of CD8α comprising SEQ ID NO: 28, (b) a transmembrane domain of CD8α comprising SEQ ID NO: 11, (c) an intracellular signaling domain of CD28 comprising SEQ ID NO: 21, and (d) an intracellular signaling domain of CD3ζ comprising SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus, (a) a hinge region of CD8α comprising SEQ ID NO: 28, (b) a transmembrane domain of CD8α comprising SEQ ID NO: 11, (c) an intracellular signaling domain of CD28 comprising SEQ ID NO: 21, and (d) an intracellular signaling domain of CD3ζ comprising SEQ ID NO: 23.

[0095] In some embodiments, a provided CAR comprises (a) an antigen binding domain comprising SEQ ID NO: 17, (b) a hinge region of CD8α comprising SEQ ID NO: 28, (c) a transmembrane domain of CD8α comprising SEQ ID NO: 11, (d) an intracellular signaling domain of CD28 comprising SEQ ID NO: 21, and (e) an intracellular signaling domain of CD3ζ comprising SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus, (a) an antigen binding domain comprising SEQ ID NO: 17, (b) a hinge region of CD8α comprising SEQ ID NO: 28, (c) a transmembrane domain of CD8α comprising SEQ ID NO: 11, (d) an intracellular signaling domain of CD28 comprising SEQ ID NO: 21, and (e) an intracellular signaling domain of CD3ζ comprising SEQ ID NO: 23.

[0096] In some embodiments, a provided CAR comprises (a) a signal peptide of CD8α comprising SEQ ID NO: 15, (b) an antigen binding domain comprising SEQ ID NO: 17, (c) a hinge region of CD8α set forth in SEQ ID NO: 28, (d) a transmembrane domain of CD8α set forth in SEQ ID NO: 11, (e) an intracellular signaling domain of CD28 set forth in SEQ ID NO: 21, and (f) an intracellular signaling domain of CD3ζ set forth in SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus, (a) a signal peptide of CD8α comprising SEQ ID NO: 15, (b) an antigen binding domain comprising SEQ ID NO: 17, (c) a hinge region of CD8α set forth in SEQ ID NO: 28, (d) a transmembrane domain of CD8α set forth in SEQ ID NO: 11, (e) an intracellular signaling domain of CD28 set forth in SEQ ID NO: 21, and (f) an intracellular signaling domain of CD3ζ set forth in SEQ ID NO: 23.

[0097] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0098] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0099] In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by the nucleic acid sequence set forth in SEQ ID NO: 14.

[0100] It has been observed that T cells engineered to express CD19-CARs incorporating the costimulatory domain of 4-1BB produce significantly higher background levels of IFNγ in the absence of CD19-expressing target cells than T cells engineered to express CD19-CARs incorporating costimulatory domains derived from CD28, CD27, or the FcεRI gamma chain. Without wishing to be bound by theory, it is hypothesized that a high background level of cytokine production is undesirable for the treatment of autoimmune diseases. Therefore, in certain embodiments, the CARs disclosed herein do not contain an intracellular T cell signaling domain derived from 4-1BB.

[0101] Nucleic Acid Constructs The present invention further provides an engineered nucleic acid or nucleic acid construct comprising a nucleic acid sequence encoding any of the polypeptides described herein, such as any of the CARs described herein. In some embodiments, the engineered nucleic acid comprises a promoter operably linked to the nucleic acid sequence encoding the CAR (e.g., any of the CARs described herein). Any suitable promoter can be operably linked to any of the engineered nucleic acid sequences described herein. Non-limiting examples of promoters that can be used in accordance with the present disclosure include EF1a, SFFV, PGK, CMV, CAG, UbC, murine stem cell virus (MSCV), MND, an EF1a hybrid promoter, a CAG hybrid promoter, or a derivative or functional fragment thereof. In some embodiments, the promoter is an EF1a promoter. In some embodiments, the promoter is an SFFV promoter. In some embodiments, the promoter is a PGK promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a UbC promoter. In some embodiments, the promoter is an MSCV promoter. In some embodiments, the promoter is an MND promoter.

[0102] In some cases, an engineered nucleic acid contains sufficient cis elements (such as a promoter and / or enhancer) to complement expression of the provided engineered nucleic acid sequence, and the remaining elements necessary for expression can be supplied by the host cell (e.g., a mammalian cell, e.g., a T cell) or an in vitro expression system.

[0103] The present disclosure also provides vectors or plasmids comprising any of the engineered nucleic acids described herein. The present disclosure provides transposons, cosmids, viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated viral (AAV) vector, lentiviral vector, and retroviral vector), and any Gateway® vector comprising any of the engineered nucleic acids described herein. In some embodiments, the vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is an AAV vector.

[0104] Exemplary lentiviral vectors that can be used in accordance with the present disclosure include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis-encephalitis virus (CAEV).

[0105] Retroviral vectors are typically constructed such that the majority of the sequences encoding the viral structural genes are deleted and replaced with a gene of interest or an expression cassette of interest (such as the engineered nucleic acids described herein). Often, the structural genes (i.e., gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques well known in the art. This involves digestion with appropriate restriction endonucleases, or optionally Bal31 exonuclease, to generate a fragment containing the appropriate portion of the packaging signal. Thus, in some embodiments, a minimal retroviral vector comprises, from 5' to 3', a 5' long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and / or enhancer, an exogenous gene of interest (or engineered nucleic acid), and a 3' LTR. In some embodiments, if no exogenous promoter is provided, gene expression can be driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. In many embodiments, the structural genes are provided in separate vectors for lentivirus production, rendering the resulting virions replication-incompetent. Specifically, in the case of lentiviruses, the packaging system can include a single packaging vector encoding the Gag, Pol, Rev, and Tat genes and a third, separate vector encoding the envelope protein Env (usually VSV-G due to its broad infectivity). To improve the safety of the packaging system, the packaging vector can be split to express Rev from one vector and Gag and Pol from another. Alternatively, Tat can be eliminated from the packaging system by using a retroviral vector containing a chimeric 5' LTR in which the U3 region of the 5' LTR has been replaced with a heterologous regulatory element.

[0106] Nucleic acids (e.g., genes) to be packaged into retroviruses (e.g., lentiviruses) can be incorporated into the proviral backbone in several general ways. The simplest configuration involves replacing the structural genes of the retrovirus with a single gene, whose transcription is under the control of viral regulatory sequences within the LTR. Retroviral vectors capable of introducing multiple genes into target cells have also been constructed. Typically, in such vectors, one gene is under the control of the viral LTR, and the second gene is expressed either from a spliced ​​message or under the control of its own internal promoter.

[0107] Thus, nucleic acids (e.g., genes) packaged into retroviruses are flanked by 5'LTR and 3'LTR, which function to promote transcription and polyadenylation of virion RNA, respectively. The term "long terminal repeat" or "LTR" refers to the base-pair domains located at the ends of retroviral DNA. These domains are direct repeats in the context of the native sequence and include the U3, R, and U5 regions. LTRs generally provide essential functions for retroviral gene expression (e.g., promotion of gene transcription, initiation, and polyadenylation) and viral replication. LTRs contain many regulatory signals, including transcription control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The U3 region contains enhancer and promoter elements. The U5 region, located between the primer binding site and the R region, contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. In certain embodiments, the R region contains a transactivation response (TAR) gene element that interacts with a transactivator (tat) gene element to promote viral replication. This element is not required in embodiments in which the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0108] In some embodiments, the retroviral vector comprises a modified 5'LTR and / or 3'LTR. Modification of the 3'LTR is often performed to render the virus replication-incompetent, thereby enhancing the safety of lentiviral or retroviral systems. In some embodiments, the retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication-incompetent retroviral vector in which the U3 region of the 3'LTR has been modified (e.g., by deletion or substitution) to prevent viral transcription after the first round of viral replication. This is because the U3 region of the 3'LTR is used as a template for the U3 region of the 5'LTR during viral replication, and therefore no viral transcripts are produced without the U3 enhancer / promoter. In some embodiments, the 3'LTR is modified so that the U5 region is replaced with, for example, an ideal polyadenylation sequence. Note that some embodiments of the present disclosure also include modifications to the LTRs, such as modifications to the 3'LTR, the 5'LTR, or both the 3'LTR and the 5'LTR.

[0109] In some embodiments, the U3 region of the 5'LTR is replaced with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include promoters from the viruses simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase). Common promoters can drive high levels of transcription in a Tat-independent manner. Because the complete U3 sequence is absent in the viral production system, this substitution reduces the likelihood of recombination resulting in replicative virus.

[0110] Adjacent to the 5'LTR are the sequence required for reverse transcription of the genome (tRNA primer binding site) and the sequence required for efficient packaging of viral RNA into particles (Psi site).As used herein, "packaging signal" or "packaging sequence" refers to the sequence located in the retroviral genome that is required for the encapsidation of retroviral RNA strands during the formation of viral particles (see, for example, Clever et al., 1995 J. Virology, 69(4):2101-09).The packaging signal can be the minimal packaging signal (also referred to as the Psi [Ψ] sequence) required for encapsidation of the viral genome.

[0111] In some embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a FLAP element. As used herein, the term "FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al. (2000) Cell 101:173. Upon reverse transcription, the central initiation of the positive-strand DNA at the cPPT and the central termination at the CTS form a triple-stranded DNA structure, i.e., a central DNA flap. Without being bound by any theory, the DNA flap is believed to act as a cis-acting determinant for the nuclear import of the lentiviral genome and / or increase viral titer. In some embodiments, the retroviral vector backbone comprises one or more FLAP elements upstream or downstream of a heterologous gene of interest within the vector. For example, in some embodiments, a transfer plasmid comprises a FLAP element. In some embodiments, a vector of the present disclosure comprises a FLAP element isolated from HIV-1.

[0112] In some embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a nuclear export element. In some embodiments, the retroviral vector comprises one or more nuclear export elements. The term "nuclear export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA nuclear export elements include the human immunodeficiency virus (HIV) RRE (see, e.g., Cullen et al., (1991) J. Virol. 65: 1053; Cullen et al., (1991) Cell 58: 423, etc.) and the hepatitis B virus post-transcriptional regulatory element (HPRE). The RNA nuclear export element is usually located within the 3'UTR of a gene and can be inserted as one or more copies.

[0113] In some embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a posttranscriptional regulatory element. Various posttranscriptional regulatory elements can increase the expression of heterologous nucleic acids, for example, the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; see Zufferey et al., (1999) J. Virol., 73:2886), the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864), the optimized posttranscriptional regulatory element (oPRE; see Schambach et al., (2006) Gene Therapy 13, 641-45), and the like (Liu et al., (1995), Genes Dev., 9:1766). The posttranscriptional regulatory element is generally located at the 3' end of the heterologous nucleic acid sequence. This configuration results in the synthesis of an mRNA transcript that contains a heterologous nucleic acid coding sequence at its 5' portion and a post-transcriptional regulatory element sequence at its 3' portion. In some embodiments, the vectors of the present disclosure lack or do not contain post-transcriptional regulatory elements such as a WPRE or HPRE, because in some cases, these elements increase the risk of cell transformation and / or do not greatly or significantly increase the amount of mRNA transcript or increase mRNA stability. Therefore, in certain embodiments, the vectors of the present disclosure lack or do not contain a WPRE or HPRE as an additional safety measure.

[0114] Elements that induce efficient termination and polyadenylation of heterologous nucleic acid transcription increase heterologous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. Thus, in some embodiments, retroviral vectors (e.g., lentiviral vectors) further comprise a polyadenylation signal. As used herein, the term "polyadenylation signal" or "polyadenylation sequence" refers to a DNA sequence that induces both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase H. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking a polyadenylation signal are unstable and rapidly degraded. Illustrative examples of polyadenylation signals that can be used in the vectors of the present disclosure include ideal polyadenylation sequences (e.g., AATAAA, ATTAAA, AGTAAA), the bovine growth hormone polyadenylation sequence (BGHpA), the rabbit β-globin polyadenylation sequence (rβgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.

[0115] In some embodiments, the retroviral vector further comprises an insulator element, which is believed to help protect sequences expressed by the retrovirus (e.g., therapeutic genes) from integration site effects (i.e., position effects, e.g., Burgess-Beusse et al., (2002) Proc. Natl. Acad. Sci., USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109:471), which are mediated by cis-elements present in the genomic DNA and can result in dysregulated expression of the introduced sequence. In some embodiments, the retroviral vector comprises an insulator element within one or both LTRs or elsewhere within the region of the vector that integrates into the cellular genome. Insulators suitable for use in the present disclosure include, but are not limited to, the chicken beta-globin insulator (see Chung et al., (1993). Cell 74:505; Chung et al., (1997) Proc. Natl. Acad. Sci., USA 94:575; and Bell et al., 1999. Cell 98:387). Examples of insulator elements include, but are not limited to, insulators from the beta-globin locus, such as chicken HS4.

[0116] Non-limiting examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech Catalog No. 631984), pLVX-EF1alpha-IRES-mCherry (Clontech Catalog No. 631987), pLVX-Puro (Clontech Catalog No. 632159), pLVX-IRES-Puro (Clontech Catalog No. 632186), pLenti6 / V5-DEST™ (Thermo Fisher), pLenti6.2 / V5-DEST™ (Thermo Fisher), and pLVX-EF1alpha-IRES-mCherry (Clontech Catalog No. 631987). Fisher), pLKO.1 (Addgene plasmid number 10878), pLKO.3G (Addgene plasmid number 14748), pSico (Addgene plasmid number 11578), pLJM1-EGFP (Addgene plasmid number 19319), FUGW (Addgene plasmid number 14883), pLVTHM (Addgene plasmid number 12247), pLVUT-tTR-KRAB (Addgene plasmid number 11651), pLL3.7 (Addgene plasmid number 11795), pLB (Addgene plasmid number 11619), pWPXL (Addgene plasmid number 12257), pWPI (Addgene plasmid number 12254), EF.CMV.RFP (Addgene plasmid number 17619), pLenti CMV Pur Examples include DEST (Addgene plasmid no. 17452), pLenti-puro (Addgene plasmid no. 39481), pULTRA (Addgene plasmid no. 24129), pLX301 (Addgene plasmid no. 25895), pHIV-EGFP (Addgene plasmid no. 21373), pLV-mCherry (Addgene plasmid no. 36084), pLionII (Addgene plasmid no. 1730), and pInducer10-mir-RUP-PheS (Addgene plasmid no. 44011). These vectors can be modified for therapeutic use.For example, the selectable marker (e.g., puro, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest. Further examples of lentiviral vectors are described in U.S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, Nos. 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, and 6,352,694, and PCT Publication No. WO2017 / 091786.

[0117] In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence encoding a CAR, wherein the nucleic acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence set forth in SEQ ID NO: 14.

[0118] In certain embodiments, the lentiviral vectors disclosed herein comprise a truncated 5'LTR (e.g., with its U3 region deleted), an HIV-1 Ψ packaging sequence, an MSCV promoter operably linked to a nucleic acid encoding a CAR (e.g., any of the CARs disclosed herein), and a truncated 3'LTR (e.g., with its U3 region deleted). In certain embodiments, the lentiviral vector further comprises an RRE, a cPPT / CTS, and / or an oPRE. In certain embodiments, the lentiviral vector comprises a truncated 5'LTR (e.g., with its U3 region deleted), an HIV-1 Ψ packaging sequence, an RRE, a cPPT / CTS, an MSCV promoter operably linked to a nucleic acid encoding a CAR (e.g., any of the CARs disclosed herein), an oPRE, and a truncated 3'LTR (e.g., with its U3 region deleted). In certain embodiments, the lentiviral vector is pseudotyped with the envelope protein of VSV-G.

[0119] How to make engineered T cells Also provided herein are methods of making engineered T cells, the methods comprising introducing into a host T cell an engineered nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., any of the CARs described herein). Thus, in some embodiments, engineered T cells refer to genetically modified T cells that have been engineered to express a CAR, e.g., any of the anti-CD19 CARs provided.

[0120] In some embodiments, host T cells used to generate engineered T cells can be any T cell, such as cultured T cells (e.g., primary T cells), T cells from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or T cells from a mammal. In some embodiments, the T cells used to generate engineered T cells can be selected from naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. In some embodiments, the host T cells used to generate engineered T cells can be CD3+ cells. In some embodiments, the host T cells can be CD4+, CD8+, or CD4+ and CD8+. In some embodiments, host T cells can be any type of T cell, such as, for example, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 cells and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), memory T cells, naive T cells, regulatory T cells, γδ T cells, etc. In some embodiments, host T cells used to generate engineered T cells can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells), etc. In some embodiments, the resulting host T cells are substantially free of non-T cells.

[0121] T cells can be obtained from a variety of biological samples from a subject (e.g., a human subject). Non-limiting examples of biological samples include cells, tissue (e.g., tissue obtained by biopsy), blood, serum, plasma, or any sample obtained therefrom. In certain embodiments, the sample is a whole blood sample or an apheresis (e.g., leukapheresis) sample taken from the subject. In certain embodiments, the method includes obtaining the biological sample from the subject. In certain embodiments, the method further includes obtaining the biological sample from the subject.

[0122] In certain embodiments, T cells are isolated from a sample. Isolation of T cells may include first purifying T cells from a mixture of plasma, lymphocytes, platelets, red blood cells, monocytes, and granulocytes. Methods for isolating T cells from biological samples, such as whole blood samples or leukapheresis samples, are well known. Exemplary methods include leukapheresis, elutriation, density gradient centrifugation, enrichment by selection, and the like. For example, the method may include obtaining or having obtained a biological sample, such as a fresh, refrigerated, frozen, or cryopreserved leukapheresis product, or an alternative source of hematopoietic tissue, such as a whole blood sample, a bone marrow sample, or a tumor or organ biopsy or resection (e.g., a thymectomy), from an entity, such as a laboratory, hospital, or healthcare provider, and performing the isolation steps described above to generate an enriched T cell population (e.g., a starting population of T cells) suitable for expression of a heterologous protein.

[0123] Furthermore, the purity of a T cell population can be increased by using one or more selection steps, such as negative selection or positive selection. Negative selection typically involves the use of one or more substances that selectively bind to undesired cell types to remove undesired cell types from a mixed cell population in a sample, whereas positive selection typically involves the use of one or more substances that selectively bind to desired cell types to isolate a desired cell population. Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies that target surface markers unique to the cells selected by negative selection. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, using a cocktail of monoclonal antibodies that target cell surface markers present on the cells selected by negative selection. For example, to enrich CD4+ cells by negative selection, the cocktail of monoclonal antibodies can include antibodies against CD14, CD20, CDb, CD16, HLA-DR, and CD8. In contrast, a positive selection step can be used to specifically select for a desired cell type. In certain embodiments, positive selection of T cells may involve incubating a mixed population of cells comprising T cells with a CD3-binding agent (e.g., anti-CD3 antibody-conjugated beads) for a time sufficient for positive selection of the desired T cells.

[0124] In some embodiments, engineered T cells are generated using a mixture of cells (e.g., a mixture of host cells). For example, a mixture of cells can be obtained (e.g., from a subject) and an engineered nucleic acid inserted into the mixture of cells to prepare a mixture of engineered cells. In some embodiments, the mixture of cells includes a mixture of T cells (e.g., any of the T cells described herein). In some embodiments, the mixture of cells includes CD4+ T cells and / or CD8+ T cells. In some embodiments, the mixture of cells includes CD4+ T cells and CD8+ T cells. In some embodiments, the mixture of cells is obtained by enriching for CD4+ T cells and CD8+ T cells to obtain an enriched mixture of CD4+ and CD8+ cells. In certain embodiments, the mixture of cells comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 20-30%, 20-40%, 20-50%, 20-60%, 30-40%, 30-50%, or 30-60% CD8 T cells of the total T cells in the population. + T cells (e.g., CD8 + In certain embodiments, the mixture of cells comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 30-40%, 30-50%, 30-60%, or 30-70% CD4 T cells of the total T cells in the population. + T cells (e.g., CD4 + In certain embodiments, the mixture of cells further comprises CD8 + T cells (e.g., CD8 + cytotoxic T cells) and CD4 + T cells (e.g., CD4 +In some embodiments, the mixture of cells comprises CD8+ T cells and CD4+ T cells in a ratio of about 1:2. In some embodiments, the host cell or mixture of host cells is expanded prior to introduction of the engineered nucleic acid, or a vector or plasmid containing the engineered nucleic acid. In some embodiments, the host cell or mixture of host cells is allogeneic. In some embodiments, the host cell or mixture of host cells is autologous.

[0125] In some embodiments, introducing the engineered nucleic acid (or a vector or plasmid comprising the engineered nucleic acid) into a host cell comprises contacting the host cell with a viral vector (e.g., any of the viral vectors described herein). In some embodiments, the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, a transposon, a cosmid, and an AAV vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, introducing the engineered nucleic acid (or a vector or plasmid comprising the engineered nucleic acid) into a host cell comprises the use of viral transduction. Any known method of introducing nucleic acids (including nucleic acid vectors and plasmids) into a host cell can be used in accordance with the present disclosure.

[0126] Methods for introducing nucleic acid constructs into cells (e.g., eukaryotic cells) are well known in the art. Non-limiting examples of methods that can be used to introduce engineered nucleic acids or nucleic acid constructs (e.g., vectors or plasmids containing engineered nucleic acids) into cells include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, transfection using dendrimers, transfection using cationic polymers, cell squeezing, sonoporation, optical transfection, impale infection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection. As used herein, "transformed" and "transduced" are used interchangeably.

[0127] In some embodiments, the engineered nucleic acid is introduced into cells using a lentiviral vector. In some embodiments, a lentiviral vector is used at a multiplicity of infection (MOI) of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more. In some embodiments, a lentiviral vector is used at an MOI of about 1. In some embodiments, a lentiviral vector is used at an MOI of about 2. In some embodiments, a lentiviral vector is used at an MOI of about 3. In some embodiments, a lentiviral vector is used at an MOI of about 4. In some embodiments, a lentiviral vector is used at an MOI of about 5. In some embodiments, a lentiviral vector is used at an MOI of about 6. In some embodiments, a lentiviral vector is used at an MOI of about 7. In some embodiments, a lentiviral vector is used at an MOI of about 8. In some embodiments, a lentiviral vector is used at an MOI of about 9. In some embodiments, a lentiviral vector is used at an MOI of about 10.

[0128] In some embodiments, the methods provided further comprise contacting the host T cells with an effective amount of one or more CD3 stimulatory agents in the absence of a CD28 stimulatory agent under conditions that allow for stimulation of the host T cells. In other embodiments, the methods provided further comprise contacting the host T cells with an effective amount of one or more agents that activate both CD3 and CD28 (e.g., a solid surface such as a polymer nanomatrix coated with an anti-CD3 antibody and an anti-CD28 antibody) under conditions that allow for stimulation of the host T cells.

[0129] In some embodiments, the present disclosure provides methods of generating engineered T cells, the methods comprising: (a) obtaining host T cells from a subject; and (b) introducing an engineered nucleic acid (e.g., any of the engineered nucleic acids described herein) into the host T cells. In some embodiments, the methods of generating engineered T cells further comprise contacting the host T cells with an effective amount of one or more CD3 stimulatory agents in the absence of a CD28 stimulatory agent under conditions that allow for stimulation of the host T cells. In other embodiments, the methods of generating engineered T cells further comprise contacting the host T cells with an effective amount of one or more agents that activate both CD3 and CD28 (e.g., magnetic beads coated with anti-CD3 and anti-CD28 antibodies) under conditions that allow for stimulation of the host T cells. In certain embodiments, the stimulating step occurs before step (b).

[0130] In some embodiments, the disclosure provides a method of making an engineered T cell, comprising: (a) obtaining a host T cell from a subject; and (b) introducing an engineered nucleic acid (e.g., a vector comprising any engineered nucleic acid described herein, e.g., an engineered nucleic acid using any of the introduction methods provided herein) into the host T cell.

[0131] In some embodiments, the disclosure provides a method of producing engineered T cells, comprising: (a) obtaining a mixture of host T cells from a subject; and (b) introducing an engineered nucleic acid (e.g., a vector comprising any engineered nucleic acid described herein, e.g., a nucleic acid engineered using any of the introduction methods provided herein) into the mixture of host T cells. In some embodiments, the method of producing engineered T cells further comprises enriching the host T cell mixture for CD4+ T cells and CD8+ T cells prior to introducing the engineered nucleic acid into the host T cell mixture.

[0132] In some embodiments, the method of generating engineered T cells further comprises expanding the host T cells or mixture of T cells prior to introducing the engineered nucleic acid or a vector or plasmid comprising the engineered nucleic acid. In certain embodiments, the T cells are expanded after introducing the engineered nucleic acid while the nucleic acid is still in the cell culture medium. In certain embodiments, the T cells are expanded for at least 3, 4, 5, 6, 7, or 8 days in the presence of one or more cytokines, including, but not limited to, IL-2, IL-7, and / or IL-15. In certain embodiments, the T cells are expanded for at least 3, 4, 5, 6, 7, or 8 days in the presence of IL-7 and IL-15.

[0133] Also provided herein are engineered T cells generated using any of the methods described herein. The present disclosure provides engineered T cells comprising an engineered nucleic acid (e.g., any of the engineered nucleic acids described herein). In many embodiments of the present disclosure, the engineered T cells comprise an engineered nucleic acid encoding a CAR (e.g., any of the CARs described herein). In some embodiments, the present disclosure provides engineered T cells comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the present disclosure provides engineered T cells comprising an engineered nucleic acid sequence set forth in SEQ ID NO: 14.

[0134] The population of engineered T cells generated includes naive T (T), characterized as CD45RO-, CCR7+, and CD95-. N ) cells, characterized as CD45RO+ and CCR7+, and central memory T (T CM ) cells, characterized as CD45RO+ and CCR7-, and effector memory T (T EM ) cells, stem cell-like memory T (T SCM ) cells, and effector memory T cells (T EMRA Alternative characteristics of these T cell subsets include, but are not limited to, naive T (T), characterized as CD45RA+, CCR7+, and CD95-. N ) cells, characterized as CD45RA- and CCR7+, and central memory T (T CM ) cells, characterized as CD45RA- and CCR7-, and effector memory T (T EM ) cells, stem cell-like memory T (T) cells characterized as CD45RA+, CCR7+, and CD95+ SCM ) cells, and effector memory T cells (T) that re-express CD45RA, characterized as CD45RA+ and CCR7-. EMRA ) cells. In some embodiments, the population comprises at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% CD4+ T cells of all T cells in the population. In some embodiments, the population comprises at least 20%, 25%, 30%, 40%, 50%, 55%, or 60% CD8+ T cells of all T cells in the population. In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% CD4+ T cells of all T cells in the population. NIn some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, or 35% CD4+ T cells of all T cells in the population. SCM In some embodiments, the population comprises at least 10%, 15%, 20%, 25%, or 30% CD4+ T cells of all T cells in the population. CM In some embodiments, the population comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% CD4+ T cells of all T cells in the population. EM In some embodiments, the population comprises at least 5%, 10%, 15%, 20%, 25%, or 30% CD4+ T cells of all T cells in the population. EMRA In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 15%, 20%, or 25% CD8+ T cells of all T cells in the population. N In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% CD8+ T cells of all T cells in the population. SCM In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 20%, 25%, or 30% CD8+ T cells of all T cells in the population. SCM In some embodiments, the population comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD8+ T cells of all T cells in the population. EMIn some embodiments, the population comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% CD8+ T cells of all T cells in the population. TEMRA Those skilled in the art will appreciate that the proportion of particular T cells present within a population of engineered T cells may vary from patient to patient.

[0135] composition Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the engineered T cells described herein or any of the engineered nucleic acids described herein. In some embodiments, the provided pharmaceutical compositions can be formulated for intravenous administration. In some embodiments, the pharmaceutical composition can include a pharmaceutically acceptable carrier (e.g., phosphate-buffered saline).

[0136] kit Also provided herein are kits comprising any of the compositions described herein. For example, the kits can comprise any one or more of the nucleic acid constructs described herein. In other examples, the kits can comprise one or more doses of any of the engineered T cells described herein or compositions comprising any of the engineered T cells described herein. In some embodiments, the kits can include instructions for carrying out any of the methods described herein.

[0137] Treatment method Provided herein are methods and compositions for reducing the number of B cells in the tissue of a subject with an autoimmune disease. In some embodiments, the disclosure provides a method for reducing the number of B cells in the tissue of a subject with an autoimmune disease, comprising administering to the subject a therapeutically effective amount of any of the engineered T cells described herein. Also provided herein are methods for treating a subject with an autoimmune disease (e.g., a B-cell-associated autoimmune disease). In some embodiments, the disclosure provides a method for treating a subject with an autoimmune disease, comprising administering to the subject a therapeutically effective amount of engineered T cells (e.g., any of the engineered T cells described herein, e.g., anti-CD19 CART cells).

[0138] In some embodiments, the B cell-associated autoimmune disease or disorder is systemic lupus erythematosus. In some embodiments, the B cell-associated autoimmune disease or disorder is lupus nephritis. In some embodiments, the B cell-associated autoimmune disease or disorder is class III or class IV lupus nephritis. In some embodiments, the B cell-associated autoimmune disease or disorder is class III lupus nephritis. In some embodiments, the B cell-associated autoimmune disease or disorder is class IV lupus nephritis. In some embodiments, the B cell-associated autoimmune disease or disorder is class II lupus nephritis.

[0139] In some embodiments, the subject to whom a provided treatment is administered (e.g., any engineered nucleic acid, engineered T cell, or CAR provided herein) has previously been treated with a lymphocyte depleting agent (e.g., cyclophosphamide and / or fludarabine). In some embodiments, the subject to whom a provided treatment is administered has previously received standard treatment for an autoimmune disease (e.g., a B-cell-associated autoimmune disease), but the treatment was ineffective and / or caused one or more adverse side effects. For example, in some embodiments, the subject to whom a treatment provided by the present disclosure is administered has previously been treated with an immunosuppressant. In some embodiments, the subject to whom a treatment provided by the present disclosure is administered has previously been treated with a corticosteroid (e.g., a glucocorticoid such as betamethasone, dexamethasone, cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, etc.). In some embodiments, the subject to whom the treatment provided herein is administered has previously been treated with a B cell-depleting antibody (e.g., an anti-CD20 antibody such as rituximab, an anti-BAFF antibody such as belimumab). In some embodiments, the subject to whom the treatment provided herein is administered has previously been treated with a calcineurin inhibitor. For example, in some embodiments, the subject to whom the treatment provided herein is administered has previously been treated with mycophenolate mofetil.

[0140] In some embodiments, the subject to whom the treatment provided herein is administered has previously received standard treatment for lupus nephritis (e.g., an immunosuppressant), and the dose of the standard treatment is reduced (e.g., by tapering) before T cells are harvested from the subject (e.g., by any of the methods described herein) to generate engineered T cells. In some embodiments, the dose of the standard treatment is reduced at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks before the treatment provided herein is administered to the subject. In some embodiments, the dose of the standard treatment is reduced at least 6 weeks before the treatment provided herein is administered to the subject. In some embodiments, the subject continues to receive standard treatment (e.g., at a reduced dose or by maintaining only a limited number of individual therapeutic components from the original SOC treatment) while also receiving the treatment provided herein. In some embodiments, the subject does not continue to receive standard treatment while receiving the treatment provided herein.

[0141] The present disclosure recognizes that treatment regimens for autoimmune diseases or disorders generally require long-term administration or multiple treatment cycles to effectively treat the disease or disorder. The most common treatments include corticosteroids and immunosuppressants, which can be highly toxic to the subject. In some cases, these drugs suppress the subject's immune system, causing serious infections and / or side effects on the bone marrow, liver, and / or kidneys. Therefore, standard treatments, such as the combination of corticosteroids and immunosuppressants, present challenges for long-term use. In contrast, the use of engineered T cells provided herein offers the potential for long-term disease management with a single infusion. The use of engineered T cells provided herein also allows for repeated treatments (e.g., long-term administration, multiple treatment cycles, etc.) due to a low toxicity profile and subsequent reduced side effects, particularly due to the fully human nature of the scFvs. In many embodiments, the use of the engineered T cells provided results in reduced toxicity, which can then be repeated in the future as needed, as adverse effects subside over time.

[0142] In some embodiments of any of the methods described herein, the administering step comprises administering two or more doses of engineered T cells (e.g., any engineered T cells described herein). In some embodiments, the administering step comprises administering five or more doses of engineered T cells. In some embodiments, the administering step comprises administering ten or more doses of engineered T cells. In some embodiments, the doses of engineered T cells are divided so that the total dose is administered over a period of at least two, three, four, five, six, seven, or more days.

[0143] The engineered T cells used in the methods of the present disclosure can be produced by the processes disclosed herein. In some embodiments, the engineered T cells are produced by introducing into T cells an engineered nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., any CAR described herein, e.g., an anti-CD19 CAR). In some embodiments, the engineered nucleic acid further comprises a promoter operably linked to the nucleic acid sequence encoding the CAR. In some embodiments, the engineered T cells are produced by further contacting the T cells with an effective amount of one or more agents that activate CD3 and CD28 under conditions that allow stimulation of the T cells. In some embodiments, prior to the steps of producing the engineered T cells and administering the engineered T cells, T cells are obtained from a subject (e.g., an autologous or allogeneic subject). The engineered T cells can be generated or produced using any of the methods of producing engineered T cells described herein, for example, in the "Methods of Producing Engineered T Cells" section above.

[0144] In some embodiments, the present disclosure provides a method of reducing the number of B cells in a tissue of a subject having an autoimmune disease, the method comprising administering engineered T cells (e.g., any of the engineered T cells described herein). In some embodiments, the present disclosure provides a method of reducing the number of B cells in a tissue of a subject having an autoimmune disease, the method comprising administering engineered T cells, wherein the engineered T cells are anti-CD19 CAR T cells. In some embodiments, the anti-CD19 CAR T cells comprise a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CD19 CAR T cells comprise a nucleic acid set forth in SEQ ID NO: 14.

[0145] In some embodiments, the present disclosure provides a method of treating lupus nephritis, the method comprising administering an engineered T cell (e.g., any of the engineered T cells described herein). In some embodiments, the present disclosure provides a method of treating lupus nephritis, the method comprising administering an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid set forth in SEQ ID NO: 14.

[0146] In some embodiments, the present disclosure provides a method of treating class III lupus nephritis, the method comprising administering an engineered T cell (e.g., any of the engineered T cells described herein). In some embodiments, the present disclosure provides a method of treating class III lupus nephritis, the method comprising administering an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid set forth in SEQ ID NO: 14.

[0147] In some embodiments, the present disclosure provides a method of treating class IV lupus nephritis, the method comprising administering an engineered T cell (e.g., any of the engineered T cells described herein). In some embodiments, the present disclosure provides a method of treating class IV lupus nephritis, the method comprising administering an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid set forth in SEQ ID NO: 14.

[0148] In some embodiments, the present disclosure provides a method of treating class II lupus nephritis, the method comprising administering an engineered T cell (e.g., any of the engineered T cells described herein). In some embodiments, the present disclosure provides a method of treating class II lupus nephritis, the method comprising administering an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CD19 CAR T cell comprises a nucleic acid set forth in SEQ ID NO: 14.

[0149] In some embodiments, the engineered T cells provided are administered using parenteral administration (e.g., intravenous administration). The methods and compositions of the present disclosure can be administered using any suitable method.

[0150] In some embodiments, administering the methods and compositions provided herein to a subject having an autoimmune disease or disorder leads to an amelioration of one or more symptoms of the autoimmune disease or disorder in the subject. In some embodiments, administering the methods and compositions provided herein to a subject having an autoimmune disease or disorder leads to a decrease in the number, severity, or frequency of one or more symptoms of the autoimmune disease or disorder in the subject (e.g., compared to the number, severity, or frequency of one or more symptoms of the autoimmune disease or disorder in the subject before treatment with a provided method or composition). In some embodiments, a subject having an autoimmune disease administered engineered T cells described herein may experience a decrease in inflammation and / or autoantibody production.

[0151] A pharmaceutical composition comprising engineered T cells and a pharmaceutically acceptable carrier or buffer can be administered to a subject with an autoimmune disease. In some embodiments, the provided pharmaceutical compositions administered to a subject with an autoimmune disease can be formulated into an injectable form (e.g., as a solution and / or suspension). In some embodiments, a pharmaceutical composition comprising engineered T cells provided herein can further comprise phosphate-buffered saline. Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, and sodium chloride.

[0152] Effective dosages for intravenous administration to a patient (e.g., for the provided T cell compositions) may vary depending on the severity of the autoimmune disease, the age and general health of the subject, the use of excipients, the potential for coadministration with other treatments, and the judgment of the treating physician. An effective amount of engineered T cells can be any amount that reduces inflammation and autoantibody production in a subject with an autoimmune disease (e.g., by removing or reducing autoreactive B cells) without causing significant toxicity to the subject. In many embodiments, the effective dosage can also depend on the level of CAR expression in the provided engineered T cells and / or the proportion of engineered T cells in the provided compositions. In some cases, the engineered T cells can be a purified population of engineered T cells generated as described herein. In some cases, the purity of the engineered T cell population can be assessed using any suitable method, including, but not limited to, flow cytometry. In some embodiments, the purity of the engineered T cell population can be assessed by quantifying the amount of CAR-expressing T cells relative to all T cells in the population. In some cases, the population of engineered T cells administered to a subject can have a purity in the range of about 5% to about 80%, about 10% to about 80%, about 15% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 80% to about 100%, about 80% to about 90%, or about 90% to 100%. In some cases, the dosage of the therapy provided (e.g., the number of engineered T cells administered) can be adjusted based on the level of purity of the therapy.

[0153] In some embodiments, compositions (e.g., pharmaceutical compositions) of engineered T cells provided comprise about 0.1 x 10 8 , about 0.2×10 8 , about 0.3×10 8 , about 0.4×10 8, about 0.5×10 8 , about 0.6×10 8 , about 0.7×10 8 , about 0.8×10 8 , about 0.9×10 8 , about 1.0×10 8 , about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , or approximately 1.5 × 10 8 In some embodiments, compositions (e.g., pharmaceutical compositions) of engineered T cells are administered at a dose of about 0.5 x 10 engineered cells. 8 In some embodiments, compositions (e.g., pharmaceutical compositions) of engineered T cells are administered at a dose of about 1.0 x 10 engineered cells. 8 The engineered cells are administered in doses of 1000 mg / kg.

[0154] The frequency of administration of the engineered T cells can be any frequency that reduces inflammation or autoantibody production in a subject with an autoimmune disease (e.g., by removing or reducing autoreactive B cells) without causing toxicity to the subject. In some embodiments, the actual frequency of administration will vary depending on various factors, including, but not limited to, the effective amount, the duration of treatment, the use of multiple therapeutic agents, and the severity of the symptoms may require increased or decreased frequency of administration.

[0155] An effective period for administering a composition comprising anti-CD19 CAR T cells or a nucleic acid encoding same can be any period that reduces inflammation or autoantibody production in a subject with an autoimmune disease (e.g., through the removal or reduction of autoreactive B cells) without causing toxicity to the subject. In some embodiments, the effective period can vary from several days to several months. In some embodiments, an effective treatment period for administering a composition comprising engineered T cells to treat an autoimmune disease can range from about 1 month to about 5 years (e.g., about 2 months to about 5 years, about 3 months to about 5 years, about 6 months to about 5 years, about 8 months to about 5 years, about 1 year to about 5 years, about 1 month to about 4 years, about 1 month to about 3 years, about 1 month to about 2 years, about 6 months to about 4 years, about 6 months to about 3 years, or about 6 months to about 2 years). In some embodiments, an effective treatment period is at least 1 year, 2 years, 3 years, or more. In some embodiments, a subject is cured (e.g., by initiating immune reset) upon infusion of the provided treatment.

[0156] In some embodiments, the course of treatment and / or the severity of one or more symptoms associated with an autoimmune disease can be monitored. Any suitable method can be used to determine whether an autoimmune disease is being treated. For example, immunological techniques (e.g., ELISA) can be performed to determine whether the level of autoantibodies present in a subject being treated as described herein decreases after administration of engineered T cells. Remission and recurrence of the disease can be monitored by examining one or more markers of the autoimmune disease.

[0157] Any suitable autoimmune disease or disorder can be treated with the engineered T cells described herein. In some cases, the autoimmune disease or disorder is caused by the accumulation of autoantibodies and can be treated with the engineered T cells described herein.

[0158] To achieve a sustained effect, a subject can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or more doses of any of the engineered T cells described herein. In some embodiments, a subject is administered at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 doses of the engineered T cells (e.g., T cells expressing a CAR, e.g., a CAR comprising the amino acid sequence set forth in SEQ ID NO: 13).

[0159] In some embodiments, administration of the provided methods and compositions results in a decrease (e.g., at least a 1% decrease, at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 60% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, at least a 95% decrease, at least a 10 ... a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, or about a 1% reduction to about a 99% reduction, about a 1% reduction to about a 90% reduction, about a 1% reduction to about a 80% reduction, about a 1% reduction to about a 70% reduction, about a 1% reduction to about a 60% reduction, about a 1% reduction to about a 50% reduction, about a 1% reduction to about a 45% reduction, about a 1% reduction to about a 4 0% decrease, approximately 1% decrease to approximately 35% decrease, approximately 1% decrease to approximately 30% decrease, approximately 1% decrease to approximately 25% decrease, approximately 1% decrease to approximately 20% decrease, approximately 1% decrease to approximately 15% decrease, approximately 1% decrease to approximately 10% decrease, approximately 1% decrease to approximately 5% decrease, approximately 5% decrease to approximately 99% decrease, approximately 5% decrease to approximately 90% decrease, approximately 5% decrease to approximately 80% decrease, approximately 5% decrease to approximately 70% decrease, approximately 5% decrease to approximately 60% decrease, approximately 5% decrease to approximately 50% decrease, approximately 5% decrease to approximately 45% decrease, approximately 5% decrease to approximately 40% decrease, approximately 5% decrease to approximately 35% decrease, approximately 5% Reduction of about 30% to about 25% reduction, Reduction of about 5% to about 20% reduction, Reduction of about 5% to about 15% reduction, Reduction of about 5% to about 10% reduction, Reduction of about 10% to about 99% reduction, Reduction of about 10% to about 90% reduction, Reduction of about 10% to about 80% reduction, Reduction of about 10% to about 70% reduction, Reduction of about 10% to about 60% reduction, Reduction of about 10% to about 50% reduction, Reduction of about 10% to about 45% reduction, Reduction of about 10% to about 40% reduction, Reduction of about 10% to about 35% reduction, Reduction of about 10% to about 30% reduction, Reduction of about 10% to about 25% reduction,Approximately 10% decrease to approximately 20% decrease, approximately 10% decrease to approximately 15% decrease, approximately 15% decrease to approximately 99% decrease, approximately 15% decrease to approximately 90% decrease, approximately 15% decrease to approximately 80% decrease, approximately 15% decrease to approximately 70% decrease, approximately 15% decrease to approximately 60% decrease, approximately 15% decrease to approximately 50% decrease, approximately 15% decrease to approximately 45% decrease, approximately 15% decrease to approximately 40% decrease, approximately 15% decrease to approximately 35% decrease, approximately 15% decrease to approximately 30% decrease, approximately 15% decrease to approximately 25% decrease, approximately 15% decrease to approximately 20% decrease, approximately 20% decrease to approximately 99% decrease, approximately 20% decrease Approximately 90% reduction, approximately 20% reduction to approximately 80% reduction, approximately 20% reduction to approximately 70% reduction, approximately 20% reduction to approximately 60% reduction, approximately 20% reduction to approximately 50% reduction, approximately 20% reduction to approximately 45% reduction, approximately 20% reduction to approximately 40% reduction, approximately 20% reduction to approximately 35% reduction, approximately 20% reduction to approximately 30% reduction, approximately 20% reduction to approximately 25% reduction, approximately 25% reduction to approximately 99% reduction, approximately 25% reduction to approximately 90% reduction, approximately 25% reduction to approximately 80% reduction, approximately 25% reduction to approximately 70% reduction, approximately 25% reduction to approximately 60% reduction, approximately 25% reduction to approximately 50% reduction, Approximately 25% reduction to approximately 45% reduction, approximately 25% reduction to approximately 40% reduction, approximately 25% reduction to approximately 35% reduction, approximately 25% reduction to approximately 30% reduction, approximately 30% reduction to approximately 99% reduction, approximately 30% reduction to approximately 90% reduction, approximately 30% reduction to approximately 80% reduction, approximately 30% reduction to approximately 70% reduction, approximately 30% reduction to approximately 60% reduction, approximately 30% reduction to approximately 50% reduction, approximately 30% reduction to approximately 45% reduction, approximately 30% reduction to approximately 40% reduction, approximately 30% reduction to approximately 35% reduction, approximately 35% reduction to approximately 99% reduction, approximately 35% reduction to approximately 90% reduction, approximately 35% reduction Approximately 80% reduction, approximately 35% reduction to approximately 70% reduction, approximately 35% reduction to approximately 60% reduction, approximately 35% reduction to approximately 50% reduction, approximately 35% reduction to approximately 45% reduction, approximately 35% reduction to approximately 40% reduction, approximately 40% reduction to approximately 99% reduction, approximately 40% reduction to approximately 90% reduction, approximately 40% reduction to approximately 80% reduction, approximately 40% reduction to approximately 70% reduction, approximately 40% reduction to approximately 60% reduction, approximately 40% reduction to approximately 50% reduction, approximately 40% reduction to approximately 45% reduction, approximately 45% reduction to approximately 99% reduction, approximately 45% reduction to approximately 90% reduction, approximately 45% reduction to approximately 80% reduction,about a 45% to about a 70% reduction, about a 45% to about a 60% reduction, about a 45% to about a 50% reduction, about a 50% to about a 99% reduction, about a 50% to about a 90% reduction, about a 50% to about a 80% reduction, about a 50% to about a 70% reduction, about a 50% to about a 60% reduction, about a 60% to about a 99% reduction, about a 60% to about a 90% reduction, about a 60% to about a 80% reduction, about a 60% to about a 70% reduction, about a 70% to about a 90% reduction, about a 70% to about a 80% reduction, about a 80% to about a 99% reduction, about a 80% to about a 90% reduction, or about a 90% to about a 99% reduction. In some embodiments, after a significant decrease in B cells is measured in a subject's tissues (e.g., peripheral blood), the number of B cells may increase and substantially recover to normal levels (e.g., compared to the subject's level before treatment, or to the level of a similar subject not receiving treatment or receiving a different treatment, or other suitable control). In some embodiments, the number of B cells is substantially recovered to normal levels after about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the recovered B cells exhibit a sustained naive phenotypic profile, indicating immune reset and the potential for long-term functional cure of the subject. Without being bound by theory, it is believed that B cell depletion caused by CAR T cells may lead to immune reset, as evidenced by the permanent resolution of disease symptoms even in the presence of reconstituted B cell numbers.

[0160] In some embodiments, administration of the methods and compositions described herein results in a decrease (e.g., at least a 1% decrease, at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least an 80% decrease, at least an 85% decrease, at least a 90% decrease, at least a 95% decrease, or at least a 99% decrease, or a decrease from about a 1% decrease to about a 99% decrease (or any subrange of this range described herein)) of autoantibody levels in a subject with an autoimmune disease, e.g., compared to the level in the subject before treatment or compared to the level in a similar subject not receiving treatment or receiving a different treatment.

[0161] The technology of the present disclosure is further described in the following examples, which do not limit the scope of the disclosure as described in the claims. [Example]

[0162] The following examples are put forth to provide one of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made, and evaluated, and are intended solely to be illustrative of the disclosure and not to limit the scope of what the inventors regard as their invention.

[0163] Example 1. Preparation of engineered T cells containing lentiviral vectors encoding Hu19-CD828Z Hu19-CD828Z can be prepared, for example, as described in U.S. Patent No. 10,287,350. Briefly, a fully human anti-CD19 CAR was generated using the sequence of the fully human 47G4 monoclonal antibody (described in U.S. Patent Application Publication No. 2010 / 0104509). The 47G4 antibody was generated by vaccinating KM strain mice carrying a human kappa light chain transgene and a human heavy chain transchromosome. The sequences of the light and heavy chain variable regions of the 47G4 antibody were obtained from U.S. Patent Application Publication No. 2010 / 0104509. The 47G4scFv was designed to contain the following elements from 5' to 3': a CD8 signal sequence, the light chain variable region of the 47G4 antibody, a linker sequence (encoding a peptide containing the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 9)) (see Cooper et al., Blood, 101(4):1637-1644 (2003)), and the heavy chain variable region of the 47G4 antibody. Next, a DNA sequence encoding a CAR was designed to contain the following elements from 5' to 3': the above-mentioned 47G4scFv, a portion of the extracellular region and the entire transmembrane region of the human CD8 molecule, and the cytoplasmic (or intracellular) portions of the human CD28 molecule and the human CD3ζ molecule. This CAR was designated 47G4-CD828Z (SEQ ID NO: 13), and its sequence was synthesized by Invitrogen (Carlsbad, Calif.).

[0164] Expression of the Hu19-CD828Z polypeptide in cells can be achieved using any suitable method. In these experiments, T cells (CD4+ T cells, CD8+ T cells, or a cell mixture enriched for CD4+ and CD8+ T cells) are transduced with a lentivirus containing a nucleic acid sequence encoding the Hu19-CD828Z polypeptide. This lentiviral vector contains, among other regulatory elements, the MSCV promoter (see, for example, Figure 5). Lentivirus is generated in HEK293 cells according to standard protocols. The KL-h198a28z lentiviral vector system can be used to transduce T cells. KL-h198a28z is a self-inactivating (SIN) vesicular stomatitis virus (VSV)-G pseudotyped third-generation lentiviral vector expressing a human anti-CD19 chimeric antigen receptor (CAR). This lentiviral vector, KL-h198a28z, is produced using a transiently transfected HEK293T cell line with a state-of-the-art four-plasmid system. The envelope protein-encoding plasmid (pLTG1292) expresses the heterologous spike protein, VSV-G, under the control of the cytomegalovirus (CMV) promoter. The VSV-G envelope protein confers broad cell tropism for transduction of a wide range of mammalian cell types. KL-h198a28z encodes the CAR construct Hu19-CD828Z, which can be used to manufacture CAR T cells for treating patients with B cell-related disorders.

[0165] Engineered T cells can be generated from blood cells using various known methods, such as those described in Ghassemi et al., (2018) Cancer Immunol. Res. 6(9) and Mackensen et al., (2022) Nat. Med. 28:2124-32. In one example, white blood cells are collected from a patient by apheresis. The cells are enriched for CD4+ and CD8+ T cells and then activated with a CD3 and CD28 agonist, such as TransAct™, a coated polymer nanomatrix. After activation, the cells are transduced with a lentiviral vector encoding Hu19-CD828Z, KL-h198a28z, and cultured for expansion. The cells are then harvested and evaluated for viability, Hu19-CD828Z expression, T cell phenotype, and efficacy (e.g., cytotoxicity and cytokine release).

[0166] Example 2. In vitro evaluation of Hu19-CD828Z transduced cells from systemic lupus erythematosus (SLE) patients The cytolytic activity and activation of Hu19-CD828Z prepared from PBMCs of SLE patients were evaluated using human CD19 + It was evaluated against malignant B-cell cancer cells and primary B cells from autologous SLE patients.

[0167] Briefly, three batches of Hu19-CD828Z cells were generated from peripheral blood mononuclear cells (PBMCs) of SLE patients, two batches of Hu19-CD828Z cells were generated from PBMCs of healthy donors, or untransduced T cells from the same donors were cocultured overnight with target cells, either the human ALL cell line NALM6 (known to express high levels of CD19), autologous (i.e., donor-matched) primary B cells expressing CD19, and / or the human chronic myeloid leukemia (CML) cell line K562, which does not express CD19. The effector:target (E:T) ratios were 0:1–3:1 for NALM6 and K562, and 0:5–10:1 for autologous primary B cell cocultures.

[0168] Effector and target cells alone were included as negative controls. After co-incubation, the amount of cytokines in the supernatant and the number of viable target cells were quantified.

[0169] SLE-derived Hu19-CD828Z cells exhibited CD19-dependent CAR-mediated cytotoxicity, cytokine release, and proliferation. + Potent and dose-dependent cytotoxicity against human B-cell cancer cell lines (NALM6) and K562 CD19 - This was demonstrated by minimal cytotoxicity against the cell lines (Figure 1). SLE-derived Hu19-CD828Z cells from two donors (3695 and 6191) induced potent dose-dependent cytotoxicity against autologous B cells, with the level of cytotoxicity significantly higher than that observed with untransduced T cells, thus indicating that the difference in cytotoxicity was driven by the CAR (Figure 2).

[0170] All three lots of SLE-derived Hu19-CD828Z cells were transfected with Hu19-CCD828Z. + When co-cultured with NALM6 cells and autologous B cells, we demonstrated CD19+-mediated and dose-dependent production of granzyme B and cytokines IL-10, IL-13, IL-2, IL-4, IL-8, and TNFα, which are directly associated with T cell activation and / or T cell-mediated cytotoxicity, whereas CD19 - This was not observed in co-culture with K562 cells (data not shown). Specifically, as shown in Figures 3A to 3B, interferon gamma (IFNγ) was detected as a major cytokine.

[0171] To test target-dependent CAR-mediated proliferation, Hu19-CD828Z cells and the same set of untransduced T cells from SLE and HD were transduced with CD19 + and CD19 -The effector cells were co-cultured with the same set of target cells for 96 hours (as described herein). Effector cells and target cells alone were included as negative controls. After co-incubation, the number of proliferating effector cells was quantified using Cell Trace Violet (CTV) dim. CD19 expression was confirmed on NALM6 and autologous primary B cells, but not on K562 cells (data not shown). SLE-derived Hu19-CD828Z cells also demonstrated comparable CAR- and CD19-mediated proliferation to HD-derived Hu19-CD828Z cells (Figures 4A-4C).

[0172] These data demonstrate that Hu19-CD828Z-transduced PBMCs from SLE patients exhibit CAR-mediated and CD19-dependent cytotoxicity, cytokine release, and cell proliferation, as demonstrated by activity when cocultured with CD19+ cancer cells (NALM6 cells) and autologous primary B cells, but not with control CD19- cells (K562). Results from cytokine release (Figures 3A-3B) and proliferation assays (Figures 4A-4C) demonstrated distinct target-mediated responses, demonstrating comparable activity of the Hu19-CD828Z CAR construct in samples from SLE and healthy donors.

[0173] Example 3. Use of Hu19-CD828Z for the treatment of lupus nephritis This example describes a phase 1 clinical trial evaluating the safety, tolerability, and clinical activity of KYV-101 (an autologous fully human anti-CD19 CAR T-cell therapy) in adult subjects with refractory lupus nephritis (LN).

[0174] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by widespread organ damage and disease severity. Approximately 50% of SLE patients may develop nephropathy (LN). LN is a major risk factor for morbidity and mortality in SLE patients, and 10% of patients with LN ultimately develop end-stage renal disease. Histopathological classification based on renal biopsy findings can determine prognosis and treatment, but focal and diffuse LN (class III and IV, respectively) require intensive immunosuppressive intervention. Despite recent approvals for LN treatment, only approximately 30–40% of patients achieve complete renal remission (CRR) at 1 year with currently available therapies, and more effective treatments remain needed.

[0175] B cells play a critical role in the pathogenesis of SLE and LN. Autoantibodies against nuclear components (e.g., double-stranded DNA (dsDNA) and Smith (Sm) protein) produced by B cells are a hallmark of this disease and can form immune complexes that cause tissue damage in target organs such as the kidneys. Diagnostic methods have been developed to detect elevated levels of antinuclear antibodies (ANA), anti-dsDNA antibodies, and anti-Sm antibodies. For example, serological tests using enzyme-linked immunosorbent assays can be used to measure anti-dsDNA and anti-Sm levels. Furthermore, ANA titers can be measured by indirect immunofluorescence assays on human epithelial type 2 (HEP-2) cells. Efficacy data from B cell-targeted therapies to date strongly support the disease-promoting role of B cells in LN and suggest that more pronounced and sustained depletion of B cells may significantly enhance efficacy.

[0176] KYV-101 contains enriched and expanded autologous CD4+ and CD8+ T cells engineered to express a CAR targeting CD19, an antigen expressed on the surface of both normal and autoreactive B cells in patients with autoimmune disease. CD19-targeted CAR T cells harness the ability of cytotoxic T cells to directly and specifically lyse target cells, effectively depleting B cells in the circulation, lymphoid tissues, and potentially non-lymphoid tissues. The CAR used in KYV-101 is Hu19-CD828Z (see, e.g., Example 1).

[0177] Patient Selection Screening of eligible subjects will occur within 28 days prior to enrollment based on at least the following inclusion criteria:

[0178] Selection criteria: 1. Subjects must be 18 years of age or older. 2. Clinical diagnosis of SLE according to the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatism (ACR) classification criteria. 3. Active LNs determined by biopsy to be proliferative LN class III or IV according to the 2018 ISN / RPS criteria. 4. Inadequate response to conventional therapy (e.g., at least two conventional therapies).

[0179] Dosage and Administration KYV-101 is an autologous anti-CD19 CAR T cell immunotherapy. The coding sequence of KYV-101 consists of a human single-chain variable fragment (scFV) CD19 targeting domain, a CD8α hinge and transmembrane domain (CD8α hinge + TM), a CD28 cytoplasmic costimulatory domain, and a CD3ζ cytoplasmic domain. Constitutive expression of the CAR is controlled by the murine stem cell virus (MSCV) promoter. KYV-101 is a cell suspension formulated in a chemically defined freezing medium. The finished product is packaged in freezer bags and stored in the vapor phase of liquid nitrogen at or below -150°C. In this study, 300 mg / m was administered for 3 days from day -7 to day -5. 2 / day cyclophosphamide and 30 mg / m 2 A standard lymphodepleting chemotherapy regimen consisting of fludarabine / day was followed by KYV-101 anti-CD19 CAR T-cell immunotherapy at approximately 1 × 10 8 CAR+ T cells and 0.5 × 10 8 The CAR T-cell therapy will be administered intravenously as a single infusion on day 0.

[0180] To evaluate the safety and tolerability of KYV-101, 9 to 12 subjects will be enrolled in a standard 3+3 dose-escalation study. Subjects will be enrolled sequentially at the dose levels shown in Table 1, depending on dose-limiting toxicities (DLTs) observed in existing patients.

[0181] [Table 1]

[0182] Preliminary results The first patient enrolled in this study was an 18-year-old female patient diagnosed with SLE at age 9. ... 8 A patient with 100 CAR T cells (100 CAR T cells) was treated with KYV-101 and had a 56-day follow-up. The patient had class IV LNs with persistent proteinuria despite treatment with mycophenolate mofetil, cyclophosphamide, calcineurin inhibitors, rituximab, belimumab, and glucocorticoids. CAR T cell generation was successful, with a CAR expression rate of 61%, purity of 99%, and viability of 96%. After infusion, CAR T cells rapidly expanded, reaching a peak of 8.6 cells / ml on day 15.

[0183] The patient experienced grade 1 cytokine release syndrome consisting of fever on days 5 and 6, which responded to acetaminophen. There were no immune effector cell-associated neurotoxicity syndromes, dose-limiting toxicities (DLTs), or serious adverse events (AEs). After CAR T cell infusion, B cell depletion was achieved, and evidence of clinical improvement was observed (Table 2). Absolute neutrophil count, hemoglobin, and platelets returned to normal levels between days 14 and 56. C-reactive protein levels normalized by day 14. Anti-dsDNA decreased slightly but remained strongly positive, and complement levels (C3, C4) increased from low to normal. Proteinuria (UPCR) improved from 1.5 to 0.5 between days 0 and 56, and the SLEDAI-2K score improved from 22 to 8. IgA and IgM levels decreased more rapidly than IgG levels. After CAR T-cell therapy, patients discontinued immunosuppressive therapy except for prednisone 10 mg, which was discontinued on day 31.

[0184] [Table 2]

[0185] These data demonstrate that KYV-101 was well tolerated, with evidence of clinical improvement, further supporting the potential use of anti-CD19 CAR T-cell therapy in the treatment of patients with LN.

[0186] Example 4. Use of Hu19-CD828Z for the treatment of lupus nephritis This example describes a Phase 1 / 2 clinical trial evaluating the safety, tolerability, and clinical activity of KYV-101 in adult subjects with refractory lupus nephritis (LN). The KYV-101 regimen used in this clinical trial was the same as that described in Example 3.

[0187] Patient Selection Screening of eligible subjects will occur within 28 days prior to enrollment based on at least the following inclusion criteria:

[0188] Selection criteria: 1. Subjects must be 18 years of age or older. 2. Clinical diagnosis of SLE according to the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatism (ACR) classification criteria. 3. Active LNs determined by biopsy to be proliferative LN class III or IV according to the 2018 ISN / RPS criteria. 4. Inadequate response to conventional therapy.

[0189] Dosage and Administration In the Phase 1 trial, 6 to 12 subjects will be enrolled in a standard single-dose study to evaluate the safety and tolerability of KYV-101. The first three subjects will receive a 1 x 10 8 Patients will be enrolled sequentially at different doses of CAR+T cells. Safety and tolerability of KYV-101 will be evaluated.

[0190] The Phase 2 study will begin after the recommended Phase 2 dose (RP2D) is identified in Phase 1. The RP2D demonstrates acceptable safety along with sufficient T-cell proliferation, B-cell depletion, and other evidence of PD activity for at least 28 days in all treated subjects. The dose-expansion portion of Phase 2 is designed to evaluate the safety, tolerability, and clinical efficacy of KYV-101 in patients with refractory LN.

[0191] Other embodiments While the technology provided has been described above in conjunction with a detailed description thereof, the above description is intended to be illustrative, but not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0192] TIFF2026503179000004.tif212165TIFF2026503179000005.tif110165TIFF2026503179000006.tif233165TIFF2026503179000007.tif234165TIFF2026503179000008.tif233165TIFF2026503179000009.tif238165TIFF2026503179000010.tif82165

Claims

1. 1. A method of treating lupus nephritis, comprising: administering to a subject in need of treatment a therapeutically effective amount of T cells comprising a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR); The CAR comprises, from the N-terminus to the C-terminus: (a) an antigen-binding fragment of an anti-CD19 antibody; (b) a transmembrane domain, and (c) Intracellular T cell signaling domain derived from human CD3ζ Including, The method.

2. 2. The method of claim 1, wherein the lupus nephritis is class III or class IV lupus nephritis.

3. The method of claim 1 or 2, wherein the anti-CD19 antibody is a human antibody.

4. the antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain; the heavy chain variable domain comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 25, 26, and 3, respectively; The method of any one of claims 1 to 3, wherein the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

5. the antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain; the heavy chain variable domain comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; The method of any one of claims 1 to 3, wherein the light chain variable domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

6. 6. The method of claim 4 or 5, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:7, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:

8.

7. 7. The method of claim 6, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

8.

8. 8. The method of claim 7, wherein the antigen-binding fragment of the anti-CD19 antibody comprises the amino acid sequence of SEQ ID NO:

17.

9. The method according to any one of claims 1 to 8, wherein the transmembrane domain is derived from human CD8.

10. The method of any one of claims 1 to 9, wherein the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

11.

11. 11. The method of claim 10, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

11.

12. 12. The method of any one of claims 1 to 11, wherein the intracellular T cell signaling domain derived from human CD3ζ comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

23.

13. 13. The method of claim 12, wherein the intracellular T cell signaling domain derived from human CD3ζ comprises the amino acid sequence of SEQ ID NO:

23.

14. The method of any one of claims 1 to 13, wherein the CAR further comprises an intracellular T cell signaling domain derived from human CD28.

15. 15. The method of claim 14, wherein the intracellular T cell signaling domain derived from human CD28 comprises the amino acid sequence of SEQ ID NO:

21.

16. The method according to any one of claims 1 to 13, wherein the CAR does not comprise an intracellular T cell signaling domain derived from 4-1BB.

17. The method of any one of claims 1 to 16, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 10 or 13.

18. The method of any one of claims 1 to 17, wherein the vector is a lentiviral vector.

19. 19. The method of any one of claims 1 to 18, wherein the vector further comprises a U3 promoter of murine stem cell virus (MSCV) operably linked to the nucleic acid.

20. The method of any one of claims 1 to 19, wherein at least 10% of the T cells express the CAR.

21. The T cells are at least 10% CD8 ＋ The method of any one of claims 1 to 20, comprising cytotoxic T cells.

22. The T cells are at least 10% CD4 ＋ The method of any one of claims 1 to 21, comprising helper T cells.

23. 23. The method of any one of claims 1 to 22, wherein the lupus nephritis is active, biopsy-proven, proliferative lupus nephritis of class III or class IV according to the 2018 ISN / RPS criteria.

23. 24. The method of claim 23, wherein the lupus nephritis is class III, active, biopsy-proven, proliferative lupus nephritis according to the 2018 ISN / RPS criteria.

24. 24. The method of claim 23, wherein the lupus nephritis is class IV, active, biopsy-proven, proliferative lupus nephritis according to the 2018 ISN / RPS criteria.

25. The therapeutically effective amount is about 5×10 ７ ~1 x 10 ８ The method of any one of claims 1 to 24, wherein the T cells are in the range of

26. The therapeutically effective amount is about 5×10 ７ The method of any one of claims 1 to 24, wherein the T cells are

27. The therapeutically effective amount is about 1×10 ８ The method of any one of claims 1 to 24, wherein the T cells are

28. 28. The method of any one of claims 1 to 27, wherein the T cells are administered by intravenous infusion.

29. 29. The method of any one of claims 1 to 28, wherein the subject is administered a single dose of the T cells.

30. The method of any one of claims 1 to 29, wherein the subject has undergone lymphocyte depletion therapy.

31. 31. The method of any one of claims 1-30, wherein the subject has undergone minimal lymphocyte-depleting therapy, resulting in about a 50% reduction in lymphocytes in the subject compared to the subject's lymphocyte count before the minimal lymphocyte-depleting therapy.

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