Methods and compositions for treating autoimmune diseases

Abstract

The present invention relates, in general terms, to a method for treating autoimmune diseases with immunotherapy. The present invention also relates, in general terms, to a method for treating autoimmune diseases comprising administering a cyclophosphamide preconditioning regimen and subsequently administering immunotherapy. TIFF2026514435000017.tif68165

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 493,133, filed on 30 March 2023, all of which disclosures thereof are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy created on March 28, 2024, is named CBB-203WO_SL.xml and has a size of 57.9 kilobytes.

[0003] Field of Invention The present invention generally relates to methods for using immunotherapy to treat autoimmune diseases. The present invention also generally relates to lymphocyte depletion preconditioning regimens that can be administered to a subject prior to immunotherapy. [Background technology]

[0004] background According to the U.S. National Institutes of Health, more than 23.5 million people in the United States suffer from autoimmune diseases. Autoimmune diseases are a group of diseases in which the body's immune system attacks healthy cells or tissues. In autoimmune diseases in which B cells are involved in the onset or maintenance of the disease, a specific population of a patient's B cells differentiates into antibody-secreting cells that produce antibodies targeted against normal tissues and cells. While these autoantibodies are effectors of the disease, the underlying cause is the defective B cells that mistakenly differentiate into cells that secrete these pathogenic antibodies.

[0005] Current treatment options for autoimmune-mediated diseases involve systemic immunosuppression achieved with corticosteroids, immunosuppressive drugs, and biological agents. Most commonly, corticosteroids are used chronically and short-term to control the disease, acting through various mechanisms that control or downmodulate multiple inflammatory pathways. In many cases, systemic immunosuppressants, such as mycophenolates, azathioprine, and methotrexate, are added to minimize the patient's symptoms and manage anticipated relapses. Most treatments for autoimmune diseases are not curative and often require lifelong chronic administration. Furthermore, patients undergoing chronic immunosuppressive therapy are at risk of serious and life-threatening infections. Thus, there is a significant unmet medical need for these patients.

[0006] Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has become an established treatment method for various cancers. This method has been particularly successful in hematological malignancies, such as acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). Generally, CAR T-cell therapy for cancer treatment involves isolating T cells from human blood, genetically engineering the cells to express a CAR against a target antigen of interest, administering a preconditioning regimen of lymphocyte depletion chemotherapy to the patient, and subsequently administering the genetically engineered CAR T cells to the patient, thereby enabling the CAR T cells to target and destroy cancer cells expressing this antigen. Lymphocyte depletion preconditioning is considered an essential element of CAR T cell therapy for cancer patients. Such preconditioning regimens have been shown to enhance the viability, persistence, and efficacy of administered CAR T cells by increasing the levels of homeostatic cytokines such as IL-7 and IL-15, eliminating endogenous immune cells that compete for these cytokines, thereby creating a more favorable environment for the proliferation of injected cells (Nissani et al. (2021) J.Immunother.Cancer 9:e001743 (Non-patent Literature 1)).

[0007] Recently, CAR T-cell therapy has also been investigated as a treatment for treatment-resistant systemic lupus erythematosus (SLE), a B-cell-mediated autoimmune disease. Researchers reported that administration of CAR T-cell therapy induced clinical remission in 5 out of 5 patients with moderate to severe treatment-resistant SLE (Mackensen et al. (2022) Nat. Med. 28:2124-2132 (Non-patent Literature 2)). In this study, patients received 75 mg / m² prior to CAR T-cell therapy. 2 The total dose of fludarabine (25 mg / m² on days -5, -4, and -3) 2 ) and 1,000 mg / m² 2 The total dose of cyclophosphamide (1,000 mg / m² on day 3) 2The patient received a lymphocyte-depleting preconditioning regimen, including Mackensen (mentioned above). Similar findings have been reported in a case study of a patient with antisynthesis syndrome (myositis) (Muller et al. (2022) Lancet 401:815-818 (Non-patent Literature 3)). Neither study examined the necessity of preconditioning regimens in achieving favorable treatment outcomes.

[0008] While lymphocyte depletion preconditioning is understood to be a crucial step in genetically engineered T-cell therapy for cancer treatment, the degree and intensity of lymphocyte depletion preconditioning required to enable successful treatment of autoimmune diseases with T-cell therapy remain unclear. Therefore, despite the progress made to date, there is still a need for novel and effective treatment methods for autoimmune diseases. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Nissani et al. (2021) J.Immunother.Cancer 9:e001743 [Non-Patent Document 2] Mackensen et al. (2022) Nat.Med.28:2124-2132 [Non-Patent Document 3] Muller et al. (2022) Lancet 401:815-818 [Overview of the project]

[0010] Preconditioning regimens have been used in genetically engineered immunotherapy to treat cancer, but given the potential for death in patients with cancer, aggressive preconditioning regimens have been considered an acceptable risk for patients. However, autoimmune diseases generally do not pose the same imminent life-threatening risks as cancer. Consequently, preconditioning measures suitable for treating cancer may not be as suitable for treating patients with autoimmune diseases, and therefore, the risks associated with such preconditioning measures may not be acceptable for patients and their physicians. Currently, when using immunotherapy to treat autoimmune diseases, the use of lower doses of preconditioning agents has been found to be effective without the risks associated with the use of higher doses of drugs used in cancer patients. The preconditioning regimens described herein, which may utilize reduced doses of cyclophosphamide compared to standard preconditioning regimens and / or may essentially consist of one or more doses of cyclophosphamide, have surprisingly been found to reduce the levels of endogenous immune cells to a degree sufficient to enable the successful treatment of autoimmune diseases with immunotherapy, while minimizing the toxicity and risks typically associated with standard preconditioning regimens.

[0011] Accordingly, in one aspect, the present disclosure is a method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease, wherein (a) 100-1,500 mg / m² 2 The present invention provides a method comprising: (b) administering to the subject a preconditioning regimen consisting of one or more doses of cyclophosphamide per day; and (b) administering to the subject a therapeutically effective amount of genetically engineered CAR immune cells, wherein the subject receives the cyclophosphamide at least three days prior to step (b). In a particular embodiment, the preconditioning regimen is 1,000 mg / m² 2Administering to a subject a cyclophosphamide total dosage of less than. In certain embodiments, one or more doses of cyclophosphamide are 100-800 mg / m 2 / day.

[0012] In another aspect, the present disclosure provides a method of treating an autoimmune disease in a subject who needs to be treated for an autoimmune disease, the method comprising: (a) administering to the subject a conditioning regimen comprising one or more doses of cyclophosphamide at 100-800 mg / m 2 / day; and (b) administering to the subject a therapeutically effective amount of genetically engineered CAR immune cells, wherein the conditioning regimen comprises administering to the subject a cyclophosphamide total dosage of less than 1,000 mg / m 2 . In certain embodiments, the subject receives cyclophosphamide more than 3 days prior to step (b).

[0013] In another aspect, the present disclosure provides a method of treating an autoimmune disease in a subject who needs to be treated for an autoimmune disease, the method comprising: (a) administering to the subject a conditioning regimen comprising one or more doses of cyclophosphamide at 100-1,500 mg / m 2 / day; and (b) administering to the subject a therapeutically effective amount of genetically engineered CAR immune cells, wherein the genetically engineered CAR immune cells comprise a nucleic acid encoding a CAR, and the CAR comprises a heavy chain variable domain (V H1 ) comprising complementarity determining regions CDR H2 , and CDR H3 , and a light chain variable domain (V H ) comprising complementarity determining regions CDR L1 , CDR L2 , and CDR L3 , and an extracellular antigen binding site comprising the CDR L , wherein the CDR H1 , CDR H2 , and CDR H3 each comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the CDR L1 , CDR L2 , and CDRL3 The present invention provides a method comprising the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively. In certain embodiments, the CAR further comprises a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In certain embodiments, the subject receives cyclophosphamide at least 3 days prior to step (b). In certain embodiments, the preconditioning regimen is 1,000 mg / m² 2 This includes administering cyclophosphamide in a total dose of less than 100 mg / m². In certain embodiments, the dose of cyclophosphamide is 100 to 800 mg / m². 2 / day

[0014] In a particular embodiment of the method described above, one or more doses of cyclophosphamide are 1,000 mg / m². 2 / day

[0015] In a particular embodiment of any of the methods described above, one or more doses of cyclophosphamide are 750 mg / m². 2 / day, 500mg / m 2 / day, 375mg / m 2 / day, or 250mg / m² 2 / day

[0016] In certain embodiments of any of the methods described above, the subject is administered cyclophosphamide 3 to 7 days before step (b). In certain embodiments, the subject is administered cyclophosphamide 3 days before step (b).

[0017] In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering cyclophosphamide to the subject 3 and 4 days prior to step (b). In certain embodiments, one or more doses of cyclophosphamide are 500 mg / m². 2 / day, 375mg / m 2 / day, or 250mg / m² 2 / day

[0018] In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering cyclophosphamide to the subject 3, 4, and 5 days prior to step (b). In certain embodiments, the dose of cyclophosphamide is 250 mg / m². 2 / day

[0019] In certain embodiments of any of the methods described above, the subject is not administered fludarabine for five days prior to step (b). In certain embodiments, the subject is not administered fludarabine for seven days prior to step (b).

[0020] In certain embodiments of the method described above, fludarabine is not administered as part of the preconditioning regimen. In certain embodiments, the subject is not administered fludarabine.

[0021] In a particular embodiment, the preconditioning regimen is the regimen disclosed in Table 1.

[0022] In a particular embodiment of any of the methods described above, a therapeutically effective amount of genetically engineered CAR immune cells is 1 × 10⁶ 5 Individual cells / kg~1×10 8 Individual cells / kg, e.g., 1 × 10⁻⁶ 6 Individual cells / kg~1×10 7 The concentration is individual cells / kg. In certain embodiments, genetically engineered CAR immune cells express CARs that specifically bind to B cell surface antigens, such as CD19.

[0023] In certain embodiments of the methods described above, the genetically engineered CAR immune cells comprise a nucleic acid encoding a CAR, wherein (i) the CAR comprises an extracellular domain containing an antigen-binding site; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain. In certain embodiments, the antigen-binding site comprises a complementarity-determining region (CDR). H1 , CDR H2 , and CDR H3 Heavy chain variable domain (VH ) and the complementarity determination region CDR L1 , CDR L2 , and CDR L3 Light chain variable domain (V L ) and, here, the CDR H1 , CDR H2 , and CDR H3 These include the amino acid sequences of SEQ ID NOs. 1, 2, and 3, respectively, and the CDR L1 , CDR L2 , and CDR L3 These contain the amino acid sequences of SEQ ID NOs. 5, 6, and 7, respectively.

[0024] In certain embodiments of the method described above, the antigen-binding site is a humanized antigen-binding site or a fully human antigen-binding site. In certain embodiments, V H and V L Each contains an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 99.5%) identical to the amino acid sequences of SEQ ID NOs. In a particular embodiment, V H and V L These include the amino acid sequences of SEQ ID NOs. 4 and 8, respectively. In certain embodiments, the antigen-binding site is located in the scFv, where, for example, the scFv includes the amino acid sequence of SEQ ID NO. 9.

[0025] In certain embodiments, the transmembrane domain of CAR includes a CD8α chain transmembrane domain, e.g., a CD8α chain transmembrane domain containing the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the co-stimulatory domain of CAR includes a 4-1BB intracellular domain, e.g., a 4-1BB intracellular domain containing the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain of CAR includes a CD3ζ signaling domain, e.g., a CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO: 21. In certain embodiments, CAR further includes a hinge domain or linker positioned between the antigen-binding site and the transmembrane domain, e.g., a CD8α chain hinge, e.g., a CD8α chain hinge containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, CAR includes the amino acid sequence of SEQ ID NO: 23.

[0026] In certain embodiments of any of the methods described above, the autoimmune disease is an autoimmune disease selected from the group consisting of B-cell-mediated autoimmune diseases, such as systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myasthenia gravis (MG), myositis, and membranous nephropathy. In certain embodiments, the autoimmune disease is selected from the group consisting of lupus nephritis, SLE with anti-dsDNA antibodies, mucosal PV, mucocutaneous PV, MuSK-associated MG, AChR MG, anti-synthesis syndrome, dermatomyositis, and immune-mediated necrotizing myopathy. In certain embodiments, the autoimmune disease is SLE.

[0027] In certain embodiments of any of the methods described above, the CAR immune cells are CAR T cells or CAR NK cells.

[0028] In another aspect, the Disclosure relates to a method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease, the method comprising administering genetically engineered immune cells comprising nucleic acids encoding a CAR, wherein the CAR comprises (i) an extracellular domain containing an antigen-binding site that binds to CD19; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain, the antigen-binding site having a complementarity-determining region (CDR). H1 , CDR H2 , and CDR H3 Heavy chain variable domain (V H ) and the complementarity determination region CDR L1 , CDR L2 , and CDR L3 Light chain variable domain (V L ) and the said CDR H1 , CDR H2 , and CDR H3 However, each contains the amino acid sequences of Sequence ID No. 1, 2, and 3, and the CDR L1 , CDR L2 , and CDR L3 The present invention provides a method comprising the amino acid sequences of Sequence ID No. 5, 6, and 7, respectively.

[0029] In certain embodiments, the antigen-binding site is a humanized antigen-binding site or a fully human antigen-binding site. In certain embodiments, V H and V L Each contains an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 99.5%) identical to the amino acid sequences of SEQ ID NOs. For example, in a particular embodiment, V H and V L These include the amino acid sequences of SEQ ID NOs. 4 and 8, respectively. In certain embodiments, the antigen-binding site is located in the scFv, where, for example, the scFv includes the amino acid sequence of SEQ ID NO. 9.

[0030] In certain embodiments, the transmembrane domain includes a CD8α transmembrane domain, for example, a CD8α transmembrane domain containing the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the co-stimulatory domain includes a 4-1BB intracellular domain, for example, a 4-1BB intracellular domain containing the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain includes a CD3ζ signaling domain, for example, a CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the CAR further includes a hinge domain or linker positioned between the antigen-binding site and the transmembrane domain, where, for example, the hinge domain is a CD8α chain hinge, for example, a CD8α chain hinge containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 23.

[0031] In certain embodiments of the aforementioned method, the immune cells are T cells or NK cells. In certain embodiments, the immune cells are T cells, for example, helper T cells, memory T cells, αβT cells, or γδT cells. In certain embodiments, the immune cells are administered to a subject in a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier or excipient. In certain embodiments of the aforementioned method, the method administers 1 × 10⁶ genetically modified immune cells. 5 Individual cells / kg~1×10 8 Dosage in individual cells / kg, e.g., 1 × 10⁻⁶ 6 Individual cells / kg~1×10 7 This includes administering the drug to the subject at a dose of individual cells / kg.

[0032] In certain embodiments, the autoimmune disease is an autoimmune disease selected from B-cell-mediated autoimmune diseases, such as systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myasthenia gravis (MG), myositis, and membranous nephropathy. In certain embodiments, the autoimmune disease is selected from lupus nephritis, SLE with anti-dsDNA antibodies, mucosal PV, mucocutaneous PV, MuSK-associated MG, AchR MG, anti-synthesis syndrome, dermatomyositis, and immune-mediated necrotizing myopathy. In certain embodiments, the autoimmune disease is SLE.

[0033] These and other aspects and features of the present disclosure are described in the following detailed description and claims. [Brief explanation of the drawing]

[0034] The present invention can be better understood by referring to the following drawings. [Figure 1] A schematic diagram of an exemplary CD19-CAR structure is shown. [Figure 2] Figures 2A–2C show schematic diagrams summarizing the Phase I / II clinical trial of CD19-CAR T-cell therapy for the treatment of SLE described in Example 2. Figure 2A summarizes the Part A protocol of the trial to determine the tolerability and bioactivity of CD19-CAR T-cell therapy (CAB-001). Figures 2B–2C summarize the Part B protocol of the trial in which subjects with either lupus nephritis (Figure 2B) or non-renal SLE (Figure 2C) are treated for 52 weeks with either CAB-001 (Group B1) or a combination of belimumab and standard treatment as a comparison group. Subjects in Group B2 may receive CAB-001 therapy at the end of 52 weeks of belimumab therapy. [Figure 3] A schematic diagram summarizing the Phase I / II clinical trial of CD19-CAR T therapy for the treatment of myasthenia gravis, as described in Example 3, including AChR antibody-positive and negative cohorts, is shown. [Figure 4]A schematic diagram summarizing the Phase I / II clinical trial of CD19-CAR T therapy for the treatment of systemic sclerosis described in Example 4 is shown, including a severe skin cohort of subjects with skin lesions that do not meet the criteria for lung, cardiac, or renal lesions, and an organ cohort of subjects that meet the criteria for lung, cardiac, or renal lesions regardless of skin lesions. [Figure 5] A schematic diagram is shown summarizing the Phase I / II clinical trial of CD19-CAR T therapy for the treatment of myositis (idiopathic inflammatory muscle disease, IIM) as described in Example 5, including the dermatomyositis cohort ("DM"), the antisynthesis syndrome cohort ("ASyS"), and the immune-mediated necrotizing myopathy cohort ("IMNM"). [Figure 6] A schematic diagram summarizing the Phase I / II clinical trial of CD19-CAR T therapy for the treatment of SLE described in Example 6, including the lupus nephritis cohort ("LN") and the non-renal SLE cohort, is shown. [Modes for carrying out the invention]

[0035] Detailed explanation Preconditioning regimens have been used with genetically and non-genetically modified immunotherapies to treat cancer, but given the potential for death in patients with cancer, aggressive preconditioning regimens have been considered an acceptable risk for patients. However, autoimmune diseases generally do not pose the same imminent life-threatening risks as cancer. Consequently, preconditioning measures suitable for treating cancer may not be as suitable for treating patients with autoimmune diseases, and therefore, the risks associated with such preconditioning measures may not be acceptable for patients and their physicians. Currently, when using immunotherapy to treat autoimmune diseases, the use of lower doses of preconditioning agents has been found to be effective without the risks associated with the use of higher doses of drugs used in cancer patients. The preconditioning regimens described herein, which may utilize reduced doses of cyclophosphamide compared to standard preconditioning regimens and / or may essentially consist of one or more doses of cyclophosphamide, have surprisingly been found to reduce the levels of endogenous immune cells to a degree sufficient to enable the successful treatment of autoimmune diseases with immunotherapy, while minimizing the toxicity and risks typically associated with standard preconditioning regimens.

[0036] I. Definition To facilitate understanding of the present invention, numerous terms and phrases are defined below.

[0037] As used herein, the terms "a" and "an" mean "one or more" and include the plural form, unless the context is appropriate.

[0038] When the term "about" is used before a quantitative value, unless otherwise specified, the present invention also includes the specific quantitative value itself. As used herein, the term "about" refers to a variation of ±10% from a baseline value, unless otherwise specified or implied.

[0039] As used herein, the term “antigen-binding site” refers to the antigen-binding fragment of an immunoglobulin or its derivative or variant that is involved in antigen binding. For example, in human antibodies, the antigen-binding site is formed by the N-terminal variable domains of the heavy and light chains, which are also referred to as the “heavy chain variable domain (VH)” and the “light chain variable domain (VL),” respectively. Within each variable domain, three highly variable stretches, referred to as “hypervariable regions,” are located between more conserved adjacent stretches known as “framework regions” (FR). The three hypervariable regions of VH and the three hypervariable regions of VL are positioned relative to each other in three-dimensional space to form a complementary antigen-binding surface on the three-dimensional surface of the antigen to be bound. The hypervariable regions are also referred to as “complementarity-determining regions” or “CDRs.” The boundary between FR and CDR can be defined using any appropriate rule known in the art, for example, the IMGT rule (see Lefranc, (1999) The Immunologist, 7, 132-136), the Kabat rule (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), or the Chothia rule (see Chothia, C. et al. (1987) J.Mol.Biol.196:901-917). Three CDRs, designated CDR1, CDR2, and CDR3, contribute to antibody binding specificity.

[0040] Examples of antigen-binding fragments of immunoglobulins include, for example, Fab, Fab', and F(ab')2 fragments. Examples of variants of antigen-binding fragments of immunoglobulins include, for example, single-chain antibodies or scFv. Certain animals have different types of antibodies. For example, camelids have V H The antibody contains the H fragment, and cartilaginous fish are V NARThe present invention relates to an antibody containing a fragment referred to as an "immunoglobulin novel antigen receptor" or "IgNAR," where such fragment is a single monomeric antibody variable domain capable of selectively binding to a specific antigen independently of another variable domain, and is referred to as a "single-domain antibody," "sdAb," or "nanobody." The antigen-binding site may contain a pair of VH and VL or either an sdAb. The antigen-binding sites disclosed herein may be recombinant, chimeric, demimmunized, humanized, and / or affinity-matured (see, for example, U.S. Patent No. 4,816,567, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-55, Morrison et al. (1985) Proc. Natl. Acad. Sci. USA, 81:6851, and Takeda et al. (1985) Nature, 314:452).

[0041] As used herein, the term “cross-competition” means, in the context of an antibody of interest and a reference antibody, that the antibody of interest competes with the reference antibody for binding to an antigen (e.g., CD19), and vice versa. The antibody of interest cross-competes with the reference antibody if competition is observed, regardless of whether the reference antibody is used as the first antibody and the antibody of interest is used as the second antibody, or whether the antibody of interest is used as the first antibody and the reference antibody is used as the second antibody. Those skilled in the art can select the concentration of the antibody to be used in the competitive assay based on the antibody’s affinity for the antigen and the antibody’s binding titer. In an exemplary assay, a first anti-CD19 antibody is immobilized on a solid surface, CD19 is bound to the first antibody, and the binding of a second anti-CD19 antibody is evaluated. If the second antibody does not produce a significant binding signal, the second antibody competes with the first antibody for binding to CD19. Exemplary assays are described in Cox et al., “Immunoassay Methods,” in Assay Guidance Manual [Internet], updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015), Silman et al. (2001) Cytometry, 44:30-37, and Finco et al. (2011) J.Pharm.Biomed.Anal., 54:351-358. CD19 conjugates containing antigen-binding sites, such as fragments of anti-CD19 antibodies or scFv derived from anti-CD19 antibodies, can also be evaluated in this assay.

[0042] The doses described herein may be presented as "body weight-based doses" or "body surface area (BSA)-based doses." Body weight-based doses are the dose administered to a patient (e.g., mg / kg) calculated based on the patient's body weight. BSA-based doses are the dose administered to a patient (e.g., mg / m²) calculated based on the patient's surface area. 2) The two forms of dose measurement for human administration can be converted by multiplying the weight-based dose by 37 or dividing the BSA-based dose by 37. For example, a dose of 60 mg / kg administered to a human subject is equivalent to 2220 mg / m³ of the same drug administered to the same subject. 2 It is equal to the dosage of [the specified number].

[0043] As used herein, the term “effective dose” means an amount of a compound or agent (e.g., the compound or agent of this disclosure) sufficient to achieve a beneficial or desired outcome. For example, “effective dose” may mean an amount of an active agent (e.g., cyclophosphamide or CAR immune cells) sufficient to achieve a beneficial or desired outcome. For example, an effective dose of an agent may be an amount sufficient to achieve one or more of the following: (1) treatment of an autoimmune disease, e.g., SLE, pemphigus vulgaris, or myositis; (2) reduction or elimination of circulating B cells in the subject; (3) reduction of autoantibody levels in the subject; (4) reduction or elimination of endogenous lymphocytes in the subject; (5) reduction of proteinuria in the subject; or (6) enhancement of the amount or activity of one or more complement factors in the subject. An effective dose may be administered in one or more doses, applications, or orally and is not intended to be limited to a particular formulation or route of administration.

[0044] As used herein, the "identity" percentage between a polypeptide sequence and a reference sequence is defined as the percentage (%) of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum possible sequence identity percentage. Similarly, the "identity" percentage between a nucleic acid sequence and a reference sequence is defined as the percentage (%) of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum possible sequence identity percentage. Alignment aimed at determining sequence identity percentages (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. A person skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

[0045] The use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, is generally open-ended and non-restrictive, and should be understood not to exclude any additional unspecified elements or steps unless specifically stated or understood otherwise from the context.

[0046] As used herein, the term “isolated” is understood to mean, when used with a particular article (e.g., polypeptide, nucleic acid, or cell), (1) that the article has been separated or purified from other components (e.g., other proteins, peptides, nucleic acids, cells, or cellular material) and / or chemicals (e.g., reagents used in its manufacture); (2) that the article can be separated or purified from an environment in which it may exist in nature, e.g., tissue or liquid sample; or (3) that the article does not exist in nature. For example, a molecule that has been taken out from the cell that produces it is “isolated.” A chemically synthesized molecule is “isolated.” As used herein, the term “isolated” may also mean a molecule that does not substantially contain other molecules of the same species. For example, a protein can be “isolated” from other proteins having different amino acid sequences. The purity or homogeneity of the article of interest may be analyzed using techniques well known in the art, including gel electrophoresis, high-performance liquid chromatography, or mass spectrometry. Any of the polynucleotides, polypeptides, vectors, compounds, or cells described herein may be isolated.

[0047] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic response, or other problems or complications, and that is commensurate with a reasonable benefit-to-risk ratio.

[0048] As used herein, the term “pharmaceutically acceptable carrier” refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that offer a reasonable benefit-risk ratio. Examples of pharmaceutically acceptable carriers include standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020).

[0049] As used herein, the term “purified” means that an entity or substance has been separated from one or more other entities or substances that have already been found to be present together with it before it is purified. Entity or substance may be partially purified, substantially purified, or pure. A substance or entity, such as a nucleic acid or polypeptide, is considered pure if it has been removed from substantially all other compounds or entities other than the solvent and any ions contained in the solvent, i.e., if it constitutes at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the dry weight of the composition. A partially or substantially purified compound or entity, such as a nucleic acid or polypeptide, may have been removed from at least 50% by weight, at least 60% by weight, at least 70% by weight, or at least 80% by weight of substances in which it is found to be present together with it, such as cellular material, such as cellular proteins and / or nucleic acids. In certain embodiments, the purified nucleic acid or polypeptide constitutes at least 10% by dry weight, 20% by dry weight, 30% by dry weight, 40% by dry weight, 50% by dry weight, 60% by dry weight, 70% by dry weight, 80% by dry weight, 90% by dry weight, 95% by dry weight, 99% by dry weight, or more of the total nucleic acid or polypeptide in the composition. Methods for evaluating purity are known in the art and include chromatography, immunological methods, and gel electrophoresis. Any of the polynucleotides, polypeptides, or cells described herein may be purified.

[0050] As used herein, the terms “subject” and “patient” refer to an organism treated by any of the methods and compositions described herein. Preferably, such organisms include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably, humans.

[0051] As used herein, “to treat,” “to treat,” and “treatment” refer to the treatment of a subject, for example, a disease, disorder, or its symptoms or signs in a human being. This includes (a) preventing a disease or disorder; (b) suppressing a disease, disorder, etc., i.e., delaying or suppressing its progression or onset; and (c) alleviating a disease, disorder, etc., i.e., causing regression of the condition. As used herein, “prevent,” “prevent,” and “prevention” refer to preventing the development of a disease, disorder, or its symptoms or signs in at least some subjects for at least a certain period of time.

[0052] II. Method In one embodiment, a method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease is disclosed herein, comprising administering to the subject a preconditioning regimen for immunotherapy, which includes or comprises one or more doses of cyclophosphamide, and subsequently administering to the subject an effective amount of genetically engineered immune cells (e.g., CAR immune cells, e.g., CAR T cells). In a related embodiment, the Disclosure provides a method for conditioning a subject for immunotherapy (e.g., CAR T cell therapy), comprising administering to the subject a preconditioning regimen, which includes or comprises one or more doses of cyclophosphamide. In another embodiment, the Disclosure provides a method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease, comprising administering to the subject an effective amount of CAR T cells, which include a CD19-specific CAR, e.g., nucleic acid encoding a CAR as described in Section III below.

[0053] a. Preconditioning regimen Cyclophosphamide is a prodrug used in conditioning regimens for the treatment of various diseases, disorders, and cancer. After administration to a subject, cyclophosphamide is known to be activated by the hepatic cytochrome P-450 enzyme system, which forms the intermediate metabolite 4-hydroxycyclophosphamide and its tautomer, aldosphamide. These two intermediate metabolites can diffuse into cells, after which aldosphamide is degraded intracellularly. In some cases, aldosphamide is converted to two molecules by β-elimination: the cytotoxic metabolite phosphoramide mustard and the by-product acrolein. Alternatively, aldosphamide can be converted to the inactive, non-toxic metabolite carboxyphosphamide by the activity of the aldehyde dehydrogenase (ALDH) enzyme. Therefore, the formation of non-toxic carboxyphosphamides is more likely to occur in cells with high levels of ALDH, such as hematopoietic stem cells and hepatocytes, while the formation of toxic metabolites, phosphoramide mustard and acrolein, is more likely to occur in cells with low levels of ALDH, such as mature B cells and T cells. Phosphoramide mustard is a bifunctional DNA alkylating molecule that reacts, for example, with the N7 atom in the imidazole ring of guanine nucleic acid bases in DNA, thereby forming covalent bonds, for example, between two guanine residues on the same DNA strand ("intra-strand crosslinks") or between two guanine residues on different DNA strands ("inter-strand crosslinks"). The formation of these DNA crosslinks interferes with DNA replication, leading to cell death. Acrolein, a byproduct of phosphoramide mustard formation, is also toxic and can cause hemorrhagic cystitis (Emadi et al. (2009) Nat. Rev. Clin. Oncol. 6:638-647).

[0054] Cyclophosphamide is used in the treatment of various indications, including cancer, such as lymphoma, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian cancer, eye cancer, and breast cancer. Cyclophosphamide is used not only in the treatment of certain autoimmune diseases, but also in conditioning regimens for allogeneic bone marrow transplantation and adoptive cell therapy for the treatment of certain cancers.

[0055] In certain embodiments, the present disclosure provides a method of treating an autoimmune disorder in a subject that needs to treat an autoimmune disorder, comprising: (a) administering to the subject a preconditioning regimen comprising one or more doses of cyclophosphamide at 100 - 2,000 mg / m 2 / day, or consisting essentially of the same; and (b) after step (a), administering to the subject a therapeutically effective amount of genetically engineered immune cells (e.g., CAR immune cells, e.g., CAR T cells).

[0056] In certain embodiments, the preconditioning regimen of the present disclosure comprises one or more doses of cyclophosphamide, or consists essentially of the same, wherein the one or more doses of cyclophosphamide are at 100 - 2,000 mg / m 2 / day. For example, in certain embodiments, the preconditioning regimen is at 100 - 2,000 mg / m 2 / day, 100 - 1,750 mg / m 2 / day, 100 - 1,500 mg / m 2 / day, 100 - 1,400 mg / m 2 / day, 100 - 1,300 mg / m 2 / day, 100 - 1,200 mg / m 2 / day, 100 - 1,100 mg / m 2 / day, 100 - 1,000 mg / m 2 / day, 100 - 950 mg / m 2 / day, 100 - 900 mg / m 2 / day, 100 - 850 mg / m 2 / day, 100 - 800 mg / m 2 / day, 100 - 750 mg / m2 / day, 100~700mg / m 2 / day, 100~650mg / m 2 / day, 100~600mg / m 2 / day, 100~550mg / m 2 / day, 100~500mg / m 2 / day, 100~450mg / m 2 / day, 100~400mg / m 2 / day, 100~375mg / m 2 / day, 100~350mg / m 2 / day, 100~300mg / m 2 / day, 100~250mg / m 2 / day, 100~200mg / m 2 / day, 100~150mg / m 2 / day, 150~2,000mg / m 2 / day, 150~1,750mg / m 2 / day, 150~1,500mg / m 2 / day, 150~1,400mg / m 2 / day, 150~1,300mg / m 2 / day, 150~1,200mg / m 2 / day, 150~1,100mg / m 2 / day, 150~1,000mg / m 2 / day, 150~950mg / m 2 / day, 150~900mg / m 2 / day, 150~850 mg / m 2 / day, 150~800mg / m 2 / day, 150~750mg / m 2 / day, 150~700mg / m 2 / day, 150~650mg / m 2 / day, 150~600mg / m 2 / day, 150~550mg / m 2 / day, 150~500mg / m 2 / day, 150~450mg / m 2 / day, 150~400mg / m 2 / day, 150~375mg / m 2 / day, 150~350mg / m 2 / day, 150~300mg / m 2 / day, 150~250mg / m 2 / day, 150~200mg / m 2 / day, 200~2,000mg / m 2 / day, 200~1,750mg / m 2 / day, 200~1,500mg / m 2 / day, 200~1,400mg / m 2 / day, 200~1,300mg / m 2 / day, 200~1,200mg / m 2 / day, 200~1,100mg / m 2 / day, 200~1,000mg / m 2 / day, 200~950mg / m 2 / day, 200~900mg / m 2 / day, 200~850mg / m 2 / day, 200~800mg / m 2 / day, 200~750mg / m 2 / day, 200~700mg / m 2 / day, 200~650mg / m 2 / day, 200~600mg / m 2 / day, 200~550mg / m 2 / day, 200~500mg / m 2 / day, 200~450mg / m 2 / day, 200~400mg / m 2 / day, 200~375mg / m 2 / day, 200~350mg / m 2 / day, 200~300mg / m 2 / day, 200~250mg / m 2 / day, 250~2,000mg / m 2 / day, 250~1,750mg / m 2 / day, 250~1,500mg / m 2 / day, 250~1,400mg / m 2 / day, 250~1,300mg / m 2 / day, 250~1,200mg / m 2 / day, 250~1,100mg / m 2 / day, 250~1,000mg / m 2 / day, 250~950mg / m 2 / day, 250~900mg / m2 / day, 250~850mg / m 2 / day, 250~800mg / m 2 / day, 250~750mg / m 2 / day, 250~700mg / m 2 / day, 250~650mg / m 2 / day, 250~600mg / m 2 / day, 250~550mg / m 2 / day, 250~500mg / m 2 / day, 250~450mg / m 2 / day, 250~400mg / m 2 / day, 250~375mg / m 2 / day, 250~350mg / m 2 / day, 250~300mg / m 2 / day, 300~2,000mg / m 2 / day, 300~1,750mg / m 2 / day, 300~1,500mg / m 2 / day, 300~1,400mg / m 2 / day, 300~1,300mg / m 2 / day, 300~1,200mg / m 2 / day, 300~1,100mg / m 2 / day, 300~1,000mg / m 2 / day, 300~950mg / m 2 / day, 300~900mg / m 2 / day, 300~850mg / m 2 / day, 300~800mg / m 2 / day, 300~750mg / m 2 / day, 300~700mg / m 2 / day, 300~650mg / m 2 / day, 300~600mg / m 2 / day, 300~550mg / m 2 / day, 300~500mg / m 2 / day, 300~450mg / m 2 / day, 300~400mg / m 2 / day, 300~375mg / m 2 / day, 300~350mg / m 2 / day, 350~2,000mg / m 2 / day, 350~1,750mg / m 2 / day, 350~1,500mg / m 2 / day, 350~1,400mg / m 2 / day, 350~1,300mg / m 2 / day, 350~1,200mg / m 2 / day, 350~1,100mg / m 2 / day, 350~1,000mg / m 2 / day, 350~950mg / m 2 / day, 350~900mg / m 2 / day, 350~850mg / m 2 / day, 350~800mg / m 2 / day, 350~750mg / m 2 / day, 350~700mg / m 2 / day, 350~650mg / m 2 / day, 350~600mg / m 2 / day, 350~550mg / m 2 / day, 350~500mg / m 2 / day, 350~450mg / m 2 / day, 350~400mg / m 2 / day, 350~375mg / m 2 / day, 375~2,000mg / m 2 / day, 375~1,750mg / m 2 / day, 375~1,500mg / m 2 / day, 375~1,400mg / m 2 / day, 375~1,300mg / m 2 / day, 375~1,200mg / m 2 / day, 375~1,100mg / m 2 / day, 375~1,000mg / m 2 / day, 375~950mg / m 2 / day, 375~900mg / m 2 / day, 375~850mg / m 2 / day, 375~800mg / m 2 / day, 375~750mg / m 2 / day, 375~700mg / m 2 / day, 375~650mg / m 2 / day, 375~600mg / m 2 / day, 375~550mg / m 2 / day, 375~500mg / m 2 / day, 375~450mg / m 2 / day, 375~400mg / m 2 / day, 400~2000mg / m 2 / day, 400~1,750mg / m 2 / day, 400~1,500mg / m 2 / day, 400~1,400mg / m 2 / day, 400~1,300mg / m 2 / day, 400~1,200mg / m 2 / day, 400~1,100mg / m 2 / day, 400~1,000mg / m 2 / day, 400~950mg / m 2 / day, 400~900mg / m 2 / day, 400~850mg / m 2 / day, 400~800mg / m 2 / day, 400~750mg / m 2 / day, 400~700mg / m 2 / day, 400~650mg / m 2 / day, 400~600mg / m 2 / day, 400~550mg / m 2 / day, 400~500mg / m 2 / day, 400~450mg / m 2 / day, 450~2,000mg / m 2 / day, 450~1,750mg / m 2 / day, 450~1,500mg / m 2 / day, 450~1,400mg / m 2 / day, 450~1,300mg / m 2 / day, 450~1,200mg / m 2 / day, 450~1,100mg / m 2 / day, 450~1,000mg / m 2 / day, 450~950mg / m 2 / day, 450~900mg / m 2 / day, 450~850mg / m 2 / day, 450~800mg / m 2 / day, 450~750mg / m2 / day, 450~700mg / m 2 / day, 450~650mg / m 2 / day, 450~600mg / m 2 / day, 450~550mg / m 2 / day, 450~500mg / m 2 / day, 500~2,000mg / m 2 / day, 500~1,750mg / m 2 / day, 500~1,500mg / m 2 / day, 500~1,400mg / m 2 / day, 500~1,300mg / m 2 / day, 500~1,200mg / m 2 / day, 500~1,100mg / m 2 / day, 500~1,000mg / m 2 / day, 500~950mg / m 2 / day, 500~900mg / m 2 / day, 500~850mg / m 2 / day, 500~800mg / m 2 / day, 500~750mg / m 2 / day, 500~700mg / m 2 / day, 500~650mg / m 2 / day, 500~600mg / m 2 / day, 500~550mg / m 2 / day, 550~2000mg / m 2 / day, 550~1,750mg / m 2 / day, 550~1,500mg / m 2 / day, 550~1,400mg / m 2 / day, 550~1,300mg / m 2 / day, 550~1,200mg / m 2 / day, 550~1,100mg / m 2 / day, 550~1,000mg / m 2 / day, 550~950mg / m 2 / day, 550~900mg / m 2 / day, 550~850mg / m 2 / day, 550~800mg / m 2 / day, 550~750mg / m 2 / day, 550~700mg / m 2 / day, 550~650mg / m 2 / day, 550~600mg / m 2 / day, 600~2000mg / m 2 / day, 600~1,750mg / m 2 / day, 600~1,500mg / m 2 / day, 600~1,400mg / m 2 / day, 600~1,300mg / m 2 / day, 600~1,200mg / m 2 / day, 600~1,100mg / m 2 / day, 600~1,000mg / m 2 / day, 600~950mg / m 2 / day, 600~900mg / m 2 / day, 600~850mg / m 2 / day, 600~800mg / m 2 / day, 600~750mg / m 2 / day, 600~700mg / m 2 / day, 600~650mg / m 2 / day, 650~2,000mg / m 2 / day, 650~1,750mg / m 2 / day, 650~1,500mg / m 2 / day, 650~1,400mg / m 2 / day, 650~1,300mg / m 2 / day, 650~1,200mg / m 2 / day, 650~1,100mg / m 2 / day, 650~1,000mg / m 2 / day, 650~950mg / m 2 / day, 650~900mg / m 2 / day, 650~850mg / m 2 / day, 650~800mg / m 2 / day, 650~750mg / m 2 / day, 650~700mg / m 2 / day, 700~2000mg / m 2 / day, 700~1,750mg / m 2 / day, 700~1,500mg / m 2 / day, 700~1,400mg / m 2 / day, 700~1,300mg / m 2 / day, 700~1,200mg / m 2 / day, 700~1,100mg / m 2 / day, 700~1,000mg / m 2 / day, 700~950mg / m 2 / day, 700~900mg / m 2 / day, 700~850mg / m 2 / day, 700~800mg / m 2 / day, 700~750mg / m 2 / day, 750~2,000mg / m 2 / day, 750~1,750mg / m 2 / day, 750~1,500mg / m 2 / day, 750~1,400mg / m 2 / day, 750~1,300mg / m 2 / day, 750~1,200mg / m 2 / day, 750~1,100mg / m 2 / day, 750~1,000mg / m 2 / day, 750~950mg / m 2 / day, 750~900mg / m 2 / day, 750~850mg / m 2 / day, 750~800mg / m 2 / day, 800~2000mg / m 2 / day, 800~1,750mg / m 2 / day, 800~1,500mg / m 2 / day, 800~1,400mg / m 2 / day, 800~1,300mg / m 2 / day, 800~1,200mg / m 2 / day, 800~1,100mg / m 2 / day, 800~1,000mg / m 2 / day, 800~950mg / m 2 / day, 800~900mg / m 2 / day, 800~850mg / m 2 / day, 850~2000mg / m 2 / day, 850~1,750mg / m 2 / day, 850~1,500mg / m 2 / day, 850~1,400mg / m 2 / day, 850~1,300mg / m 2 / day, 850~1,200mg / m 2 / day, 850~1,100mg / m 2 / day, 850~1,000mg / m 2 / day, 850~950mg / m 2 / day, 850~900mg / m 2 / day, 900~2000mg / m 2 / day, 900~1,750mg / m 2 / day, 900~1,500mg / m 2 / day, 900~1,400mg / m 2 / day, 900~1,300mg / m 2 / day, 900~1,200mg / m 2 / day, 900~1,100mg / m 2 / day, 900~1,000mg / m 2 / day, 900~950mg / m 2 / day, 950~2000mg / m 2 / day, 950~1,750mg / m 2 / day, 950~1,500mg / m 2 / day, 950~1,400mg / m 2 / day, 950~1,300mg / m 2 / day, 950~1,200mg / m 2 / day, 950~1,100mg / m 2 / day, 950~1,000mg / m 2 / day, 1,000~2,000mg / m 2 / day, 1,000~1,750mg / m 2 / day, 1,000~1,500mg / m 2 / day, 1,000~1,400mg / m 2 / day, 1,000~1,300mg / m 2 / day, 1,000~1,200mg / m 2 / day, 1,000~1,100mg / m 2 / day, 1,100~2,000mg / m 2 / day, 1,100~1,750mg / m 2 / day, 1,100~1,500mg / m 2 / day, 1,100~1,400mg / m 2 / day, 1,100~1,300mg / m 2 / day, 1,100~1,200mg / m 2 / day, 1,200~2,000mg / m 2 / day, 1,200~1,750mg / m 2 / day, 1,200~1,500mg / m 2 / day, 1,200~1,400mg / m 2 / day, 1,200~1,300mg / m 2 / day, 1,300~2,000mg / m 2 / day, 1,300~1,750mg / m 2 / day, 1,300~1,500mg / m 2 / day, 1,300~1,400mg / m 2 / day, 1,400~2,000mg / m 2 / day, 1,400~1,750mg / m 2 / day, 1,400~1,500mg / m 2 / day, 1,500~2,000mg / m 2 / day, 1,500~1,750mg / m 2 / day, 1,600~2,000mg / m 2 / day, 1,600~1,750mg / m 2 / day, or 1,750-2,000 mg / m² 2 The treatment involves, or essentially consists of, administering one or more doses of cyclophosphamide per day to the target. In certain embodiments, the one or more doses of cyclophosphamide are 200 to 1,500 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 250 to 1,000 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 250-750 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 250-500 mg / m² each. 2This is / day. In certain embodiments, one or more doses of cyclophosphamide are 250-375 mg / m² each. 2 This is per day. In certain embodiments, one or more doses of cyclophosphamide are 375 to 1,000 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 375-750 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 375-500 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 500-1,000 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 500-750 mg / m² each. 2 This is / day. In certain embodiments, one or more doses of cyclophosphamide are 750-1,000 mg / m² each. 2 / day

[0057] In certain embodiments, the preconditioning regimen of the present disclosure includes, or essentially comprises, administering one or more doses of cyclophosphamide to the target, wherein each of the one or more doses of cyclophosphamide is 100 mg / m². 2 Less than / day, 125 mg / m² 2 Less than 150 mg / m² per day 2 Less than 175 mg / m² per day, 175 mg / m² 2 Less than 200 mg / m² per day 2 Less than / day, 225 mg / m² 2 Less than 250 mg / m² per day 2 Less than / day, 275 mg / m² 2 Less than 300 mg / m² per day 2 Less than 350 mg / m² per day 2 Less than 375 mg / m² per day. 2 Less than 400 mg / m² per day 2 Less than 450 mg / m² per day 2 Less than 500 mg / m² per day 2 Less than 550 mg / m² per day 2 Less than 600 mg / m² per day2 Less than 650 mg / m² per day 2 Less than 700 mg / m² per day 2 Less than 750 mg / m² per day 2 Less than 800 mg / m² per day 2 Less than 850 mg / m² per day 2 Less than 900 mg / m² per day 2 Less than 950 mg / m² per day 2 Less than 1,000 mg / m² per day 2 Less than 1,100 mg / m² per day 2 Less than 1,200 mg / m² per day 2 Less than 1,250 mg / m² per day. 2 Less than 1,300 mg / m² per day 2 Less than 1,400 mg / m² per day 2 Less than 1,500 mg / m² per day 2 Less than 1,600 mg / m² per day 2 Less than 1,700 mg / m² per day 2 Less than 1,750 mg / m² per day. 2 Less than 1,800 mg / m² per day 2 Less than 1,900 mg / m² per day 2 Less than 2,000 mg / m² per day, or 2,000 mg / m². 2 Less than / day. In certain embodiments, the single or multiple doses of cyclophosphamide administered to the subject are 1,000 mg / m² each. 2 Less than 800 mg / m² / day. In certain embodiments, the one or more doses of cyclophosphamide administered to the subject are 800 mg / m² each. 2 The dose is less than 600 mg / m² / day. In certain embodiments, the single or multiple doses of cyclophosphamide administered to the subject are 600 mg / m² each. 2 The dose is less than 500 mg / m². In certain embodiments, the single or multiple doses of cyclophosphamide administered to the subject are 500 mg / m² each. 2 It is less than one day.

[0058] In certain embodiments, the preconditioning regimen of the present disclosure comprises, or essentially comprises, administering one or more doses of cyclophosphamide to the target, wherein each of the one or more doses of cyclophosphamide is 100 mg / m². 2 / day, 125mg / m 2 / day, 150mg / m 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, 300mg / m 2 / day, 350mg / m 2 / day, 375mg / m 2 / day, 400mg / m 2 / day, 450mg / m 2 / day, 500mg / m 2 / day, 550mg / m 2 / day, 600mg / m 2 / day, 650mg / m 2 / day, 700mg / m 2 / day, 750mg / m 2 / day, 800mg / m 2 / day, 850mg / m 2 / day, 900mg / m 2 / day, 950mg / m 2 / day, 1,000mg / m 2 / day, 1,100mg / m 2 / day, 1,200mg / m 2 / day, 1,250mg / m 2 / day, 1,300mg / m 2 / day, 1,400mg / m 2 / day, 1,500mg / m 2 / day, 1,600mg / m 2 / day, 1,700mg / m 2 / day, 1,750mg / m 2 / day, 1,800mg / m 2 / day, 1,900mg / m 2 / day, or 2,000 mg / m² 2 This is / day. In a particular embodiment, each of the one or more doses of cyclophosphamide is 250 mg / m². 2 This is / day. In a particular embodiment, each of the one or more doses of cyclophosphamide is 375 mg / m². 2 This is / day. In a particular embodiment, each of the one or more doses of cyclophosphamide is 500 mg / m².2 This is / day. In a particular embodiment, each of the one or more doses of cyclophosphamide is 750 mg / m². 2 This is / day. In a particular embodiment, each of the one or more doses of cyclophosphamide is 1,000 mg / m². 2 / day

[0059] In certain embodiments, the preconditioning regimen of the present disclosure comprises, or essentially comprises, administering one or more doses of cyclophosphamide to a subject, wherein the dose of cyclophosphamide is administered to the subject daily. In certain embodiments, the dose of cyclophosphamide is administered to the subject daily for 1, 2, 3, 4, or 5 days. In certain embodiments, the dose of cyclophosphamide is administered to the subject daily for 3 days. In certain embodiments, the dose of cyclophosphamide is administered to the subject daily for 2 days. In certain embodiments, a single dose of cyclophosphamide is administered to the subject for only 1 day.

[0060] In certain embodiments, the therapeutic methods of the present disclosure include (a) administering a preconditioning regimen comprising or essentially comprising one or more doses of cyclophosphamide to a target; and (b) administering a therapeutically effective amount of genetically engineered immune cells (e.g., CAR immune cells, e.g., CAR T cells) to a target after step (a).

[0061] In certain embodiments, the subject receives cyclophosphamide one, two, three, four, five, six, seven, eight, nine, or ten days or more before step (b) (for example, before the start of step (b)). In certain embodiments, the subject receives cyclophosphamide one, two, three, four, or five days or more before step (b) (for example, before the start of step (b)). In certain embodiments, the subject receives cyclophosphamide one, two, or three days or more before step (b) (for example, before the start of step (b)). In certain embodiments, the subject receives cyclophosphamide three days or more before step (b) (for example, before the start of step (b)).

[0062] In certain embodiments, one or more doses of cyclophosphamide are administered to the subject within 10 days prior to step (b) (for example, within 10 days prior to the start of step (b)). In certain embodiments, one or more doses of cyclophosphamide are administered to the subject within 7 days prior to step (b) (for example, within 7 days prior to the start of step (b)). In certain embodiments, one or more doses of cyclophosphamide are administered to the subject within 5 days prior to step (b) (for example, within 5 days prior to the start of step (b)). In certain embodiments, one or more doses of cyclophosphamide are administered to the subject within 4 days prior to step (b) (for example, within 4 days prior to the start of step (b)). In certain embodiments, one or more doses of cyclophosphamide are administered to the subject within 3 days prior to step (b) (for example, within 3 days prior to the start of step (b)). In certain embodiments, one or more doses of cyclophosphamide are administered to the subject within three days prior to step (b) (for example, within three days prior to the start of step (b)). In certain embodiments, each of the one or more doses of cyclophosphamide is administered to the subject 10, 9, 8, 7, 6, 5, 4, 3, 2, and / or 1 day prior to step (b) (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, and / or 1 day prior to the start of step (b)).

[0063] In certain embodiments, each of one or more doses of cyclophosphamide is administered to the subject 7, 6, 5, 4, 3, 2, and / or 1 day before step (b) (for example, 7, 6, 5, 4, 3, 2, and / or 1 day before the start of step (b)). In certain embodiments, each of one or more doses of cyclophosphamide is administered to the subject 5, 4, 3, 2, and / or 1 day before step (b) (for example, 5, 4, 3, 2, and / or 1 day before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject 5 days before step (b) (for example, 5 days before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject 2 days before step (b) (for example, 2 days before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject one day before step (b) (for example, one day before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject four days before step (b) (for example, four days before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject three days before step (b) (for example, three days before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject four and three days before step (b) (for example, four and three days before the start of step (b)). In certain embodiments, the dose of cyclophosphamide is administered to the subject five days, four days, and three days before step (b) (for example, five days, four days, and three days before the start of step (b)).

[0064] In a particular embodiment, step (b) is defined as taking place on "Day 0," where "-X Day" and "X Day" refer to a day that is X days before or X days after Day 0, respectively. In a particular embodiment, the subject receives cyclophosphamide by Day -1, Day -2, Day -3, Day -4, Day -5, Day -6, Day -7, Day -8, Day -9, or Day -10. In a particular embodiment, the subject receives cyclophosphamide by Day -2. In a particular embodiment, the subject receives cyclophosphamide by Day -3.

[0065] In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -10 and before day 0. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -10 and before day -2. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -7 and before day 0. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -7 and before day -2. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -6 and before day 0. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -6 and before day -2. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -5 and before day 0. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -5 and before day -2. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -4 and before day 0. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -4 and before day -2. In certain embodiments, one or more doses of cyclophosphamide are administered to the subject on or after day -3 and before day 0.

[0066] In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering doses of cyclophosphamide to target on day -10, day -9, day -8, day -7, day -6, day -5, day -4, day -3, day -2, and / or day -1. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering doses of cyclophosphamide to target on day -7, day -6, day -5, day -4, day -3, day -2, and / or day -1. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering doses of cyclophosphamide to target on day -5, day -4, day -3, day -2, and / or day -1. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering doses of cyclophosphamide to target on day -2. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering a dose of cyclophosphamide to target on day -1. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering a dose of cyclophosphamide to target on day -5. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering a dose of cyclophosphamide to target on day -4. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering a dose of cyclophosphamide to target on day -3. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering doses of cyclophosphamide to target on days -4 and -3. In certain embodiments, the preconditioning regimen includes, or essentially consists of, administering doses of cyclophosphamide to target on days -5, -4, and -3.

[0067] In certain embodiments, the preconditioning regimen of the present disclosure comprises, or essentially comprises, administering one or more doses of cyclophosphamide to a subject, wherein the total dose of cyclophosphamide administered to the subject is 100 to 2,000 mg / m². 2 For example, if the target is 500 mg / m² 2 If cyclophosphamide is administered at a daily dose on days -4 and -3, the subject in question will receive 1,000 mg / m². 2 The total dose of cyclophosphamide is considered to have been administered. The subject was 1,000 mg / m². 2 If a single dose of cyclophosphamide per day is administered on day 3, the subject in question should receive 1,000 mg / m². 2 A total dose of cyclophosphamide is considered to have been administered. In certain embodiments, the preconditioning regimen is 100-2,000 mg / m². 2 , 100~1,750 mg / m² 2 100-1,500 mg / m² 2 , 100~1,400 mg / m² 2 , 100~1,300 mg / m² 2 , 100~1,200 mg / m² 2 , 100~1,100 mg / m² 2 , 100~1,000 mg / m² 2 , 100-950 mg / m² 2 , 100-900 mg / m² 2 , 100-850 mg / m² 2 , 100-800 mg / m² 2 , 100-750 mg / m² 2 , 100-700 mg / m² 2 , 100-650 mg / m² 2 , 100-600 mg / m² 2 , 100-550 mg / m² 2 , 100-500 mg / m² 2 , 100-450 mg / m² 2 , 100-400 mg / m² 2 , 100-375 mg / m² 2 , 100-350 mg / m² 2 , 100-300 mg / m² 2 , 100-250 mg / m²2 、100~200mg / m 2 、100~150mg / m 2 、150~2,000mg / m 2 、150~1,750mg / m 2 、150~1,500mg / m 2 、150~1,400mg / m 2 、150~1,300mg / m 2 、150~1,200mg / m 2 、150~1,100mg / m 2 、150~1,000mg / m 2 、150~950mg / m 2 、150~900mg / m 2 、150~850mg / m 2 、150~800mg / m 2 、150~750mg / m 2 、150~700mg / m 2 、150~650mg / m 2 、150~600mg / m 2 、150~550mg / m 2 、150~500mg / m 2 、150~450mg / m 2 、150~400mg / m 2 、150~375mg / m 2 、150~350mg / m 2 、150~300mg / m 2 、150~250mg / m 2 、150~200mg / m 2 、200~2,000mg / m 2 、200~1,750mg / m 2 、200~1,500mg / m 2 、200~1,400mg / m 2 、200~1,300mg / m 2 、200~1,200mg / m 2 、200~1,100mg / m 2 、200~1,000mg / m 2 、200~950mg / m 2 、200~900mg / m 2 、200~850mg / m 2、200-800mg / m 2 、200-750mg / m 2 、200~700mg / m 2 、200~650mg / m 2 、200~600mg / m 2 、200~550mg / m 2 、200~500mg / m 2 、200~450mg / m 2 、200~400mg / m 2 、200~375mg / m 2 、200~350mg / m 2 、200~300mg / m 2 、200~250mg / m 2 、250~2,000mg / m 2 、250~1,750mg / m 2 、250~1,500mg / m 2 、250~1,400mg / m 2 、250~1,300mg / m 2 、250~1,200mg / m 2 、250~1,100mg / m 2 、250~1,000mg / m 2 、250~950mg / m 2 、250~900mg / m 2 、250~850mg / m 2 、250~800mg / m 2 、250~750mg / m 2 、250~700mg / m 2 、250~650mg / m 2 、250~600mg / m 2 、250~550mg / m 2 、250~500mg / m 2 、250~450mg / m 2 、250~400mg / m 2 、250~375mg / m 2 、250~350mg / m 2 、250~300mg / m 2 、300~2,000mg / m 2 、300~1,750mg / m 2 、300~1,500mg / m 2、300~1,400mg / m 2 、300~1,300mg / m 2 、300~1,200mg / m 2 、300~1,100mg / m 2 、300~1,000mg / m 2 、300~950mg / m 2 、300~900mg / m 2 、300~850mg / m 2 、300~800mg / m 2 、300~750mg / m 2 、300~700mg / m 2 、300~650mg / m 2 、300~600mg / m 2 、300~550mg / m 2 、300~500mg / m 2 、300~450mg / m 2 、300~400mg / m 2 、300~375mg / m 2 、300~350mg / m 2 、350~2,000mg / m 2 、350~1,750mg / m 2 、350~1,500mg / m 2 、350~1,400mg / m 2 、350~1,300mg / m 2 、350~1,200mg / m 2 、350~1,100mg / m 2 、350~1,000mg / m 2 、350~950mg / m 2 、350~900mg / m 2 、350~850mg / m 2 、350~800mg / m 2 、350~750mg / m 2 、350~700mg / m 2 、350~650mg / m 2 、350~600mg / m 2 、350~550mg / m 2 、350~500mg / m 2 、350~450mg / m 2 、350~400mg / m 2、350~375mg / m 2 、375-2,000mg / m 2 、375-1,750mg / m 2 、375-1,500mg / m 2 、375-1,400mg / m 2 、375-1,300mg / m 2 、375-1,200mg / m 2 、375-1,100mg / m 2 、375-1,000mg / m 2 、375-950mg / m 2 、375-900mg / m 2 、375-850mg / m 2 、375-800mg / m 2 、375-750mg / m 2 、375-700mg / m 2 、375-650mg / m 2 、375-600mg / m 2 、375-550mg / m 2 、375-500mg / m 2 、375-450mg / m 2 、375-400mg / m 2 、400~2,000mg / m 2 、400~1,750mg / m 2 、400~1,500mg / m 2 、400~1,400mg / m 2 、400~1,300mg / m 2 、400~1,200mg / m 2 、400~1,100mg / m 2 、400~1,000mg / m 2 、400~950mg / m 2 、400~900mg / m 2 、400~850mg / m 2 、400~800mg / m 2 、400~750mg / m 2 、400~700mg / m 2 、400~650mg / m 2 、400~600mg / m 2 、400~550mg / m 2 、400~500mg / m2 、400~450mg / m 2 、450~2,000mg / m 2 、450~1,750mg / m 2 、450~1,500mg / m 2 、450~1,400mg / m 2 、450~1,300mg / m 2 、450~1,200mg / m 2 、450~1,100mg / m 2 、450~1,000mg / m 2 、450~950mg / m 2 、450~900mg / m 2 、450~850mg / m 2 、450~800mg / m 2 、450~750mg / m 2 、450~700mg / m 2 、450~650mg / m 2 、450~600mg / m 2 、450~550mg / m 2 、450~500mg / m 2 、500~2,000mg / m 2 、500~1,750mg / m 2 、500~1,500mg / m 2 、500~1,400mg / m 2 、500~1,300mg / m 2 、500~1,200mg / m 2 、500~1,100mg / m 2 、500~1,000mg / m 2 、500~950mg / m 2 、500~900mg / m 2 、500~850mg / m 2 、500~800mg / m 2 、500~750mg / m 2 、500~700mg / m 2 、500~650mg / m 2 、500~600mg / m 2 、500~550mg / m 2 、550~2,000mg / m 2 、550~1,750mg / m 2、550~1,500mg / m 2 、550~1,400mg / m 2 、550~1,300mg / m 2 、550~1,200mg / m 2 、550~1,100mg / m 2 、550~1,000mg / m 2 、550~950mg / m 2 、550~900mg / m 2 、550~850mg / m 2 、550~800mg / m 2 、550~750mg / m 2 、550~700mg / m 2 、550~650mg / m 2 、550~600mg / m 2 、600~2,000mg / m 2 、600~1,750mg / m 2 、600~1,500mg / m 2 、600~1,400mg / m 2 、600~1,300mg / m 2 、600~1,200mg / m 2 、600~1,100mg / m 2 、600~1,000mg / m 2 、600~950mg / m 2 、600~900mg / m 2 、600~850mg / m 2 、600~800mg / m 2 、600~750mg / m 2 、600~700mg / m 2 、600~650mg / m 2 、650~2,000mg / m 2 、650~1,750mg / m 2 、650~1,500mg / m 2 、650~1,400mg / m 2 、650~1,300mg / m 2 、650~1,200mg / m 2 、650~1,100mg / m 2 、650~1,000mg / m 2 、650~950mg / m2 、650~900mg / m 2 、650~850mg / m 2 、650~800mg / m 2 、650~750mg / m 2 、650~700mg / m 2 、700~2,000mg / m 2 、700~1,750mg / m 2 、700~1,500mg / m 2 、700~1,400mg / m 2 、700~1,300mg / m 2 、700~1,200mg / m 2 、700~1,100mg / m 2 、700~1,000mg / m 2 、700~950mg / m 2 、700~900mg / m 2 、700~850mg / m 2 、700~800mg / m 2 、700~750mg / m 2 、750~2,000mg / m 2 、750~1,750mg / m 2 、750~1,500mg / m 2 、750~1,400mg / m 2 、750~1,300mg / m 2 、750~1,200mg / m 2 、750~1,100mg / m 2 、750~1,000mg / m 2 、750~950mg / m 2 、750~900mg / m 2 、750~850mg / m 2 、750~800mg / m 2 、800~2,000mg / m 2 、800~1,750mg / m 2 、800~1,500mg / m 2 、800~1,400mg / m 2 、800~1,300mg / m 2 、800~1,200mg / m 2 、800~1,100mg / m 2 、800~1,000mg / m2 、800~950mg / m 2 、800~900mg / m 2 、800~850mg / m 2 、850~2,000mg / m 2 、850~1,750mg / m 2 、850~1,500mg / m 2 、850~1,400mg / m 2 、850~1,300mg / m 2 、850~1,200mg / m 2 、850~1,100mg / m 2 、850~1,000mg / m 2 、850~950mg / m 2 、850~900mg / m 2 、900~2,000mg / m 2 、900~1,750mg / m 2 、900~1,500mg / m 2 、900~1,400mg / m 2 、900~1,300mg / m 2 、900~1,200mg / m 2 、900~1,100mg / m 2 、900~1,000mg / m 2 、900~950mg / m 2 、950~2,000mg / m 2 、950~1,750mg / m 2 、950~1,500mg / m 2 、950~1,400mg / m 2 、950~1,300mg / m 2 、950~1,200mg / m 2 、950~1,100mg / m 2 、950~1,000mg / m 2 、1,000~2,000mg / m 2 、1,000~1,750mg / m 2 、1,000~1,500mg / m 2 、1,000~1,400mg / m 2 、1,000~1,300mg / m 2 、1,000~1,200mg / m 2 、1,000~1,100mg / m2 1,100-2,000 mg / m² 2 1,100~1,750 mg / m² 2 1,100~1,500 mg / m² 2 1,100~1,400 mg / m² 2 1,100-1,300 mg / m² 2 , 1,100~1,200 mg / m² 2 1,200~2,000 mg / m² 2 1,200~1,750 mg / m² 2 1,200~1,500 mg / m² 2 1,200~1,400 mg / m² 2 1,200-1,300 mg / m² 2 1,300-2,000 mg / m² 2 1,300~1,750 mg / m² 2 1,300-1,500 mg / m² 2 1,300-1,400 mg / m² 2 1,400-2,000 mg / m² 2 1,400~1,750 mg / m² 2 1,400-1,500 mg / m² 2 1,500-2,000 mg / m² 2 1,500~1,750 mg / m² 2 , or 1,750-2,000 mg / m² 2 This includes administering a total dose of cyclophosphamide to the target population.

[0068] In certain embodiments, the total dose of cyclophosphamide is administered to the subject over one day. For example, in certain embodiments, the total dose of cyclophosphamide is administered on, for example, day -5, day -4, day -3, day -2, or day -1. In certain embodiments, the total dose of cyclophosphamide is administered on day -4. In certain embodiments, the total dose of cyclophosphamide is administered on day -3. In certain embodiments, the total dose of cyclophosphamide is administered on day -2. In certain embodiments, the total dose of cyclophosphamide is administered on day -1.

[0069] In certain embodiments, the preconditioning regimen of the present disclosure comprises or consists essentially of administering one or more doses of cyclophosphamide, wherein the total dose of cyclophosphamide administered to the subject is less than 2,000 mg / m 2 For example, in certain embodiments, the total dose of cyclophosphamide administered to the subject is less than 100 mg / m 2 less than 125 mg / m 2 less than 150 mg / m 2 less than 175 mg / m 2 less than 200 mg / m 2 less than 225 mg / m 2 less than 250 mg / m 2 less than 275 mg / m 2 less than 300 mg / m 2 less than 350 mg / m 2 less than 375 mg / m 2 less than 400 mg / m 2 less than 450 mg / m 2 less than 500 mg / m 2 less than 550 mg / m 2 less than 600 mg / m 2 less than 650 mg / m 2 less than 700 mg / m 2 less than 750 mg / m 2 less than 800 mg / m 2 less than 850 mg / m 2 less than 900 mg / m 2 less than 950 mg / m 2 less than 1,000 mg / m 2 less than 1,100 mg / m 2 less than 1,200 mg / m 2 less than 1,250 mg / m 2 less than 1,300 mg / m 2 less than 1,400 mg / m 2 less than 1,500 mg / m 2 less than 1,600 mg / m 2 less than 1,700 mg / m 2 less than 1,750 mg / m 2 less than 1,800 mg / m 2 less than 1,900 mg / m 2Less than 2,000 mg / m² 2 It is less than 2,000 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 2,000 mg / m². 2 It is less than 1,500 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 1,500 mg / m². 2 It is less than 1,000 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 1,000 mg / m². 2 It is less than 800 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 800 mg / m². 2 It is less than 600 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 600 mg / m². 2 It is less than 500 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 500 mg / m². 2 It is less than 300 mg / m². In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 300 mg / m². 2 It is less than.

[0070] In certain embodiments, the preconditioning regimen of the present disclosure comprises, or essentially comprises, administering one or more doses of cyclophosphamide to a subject, wherein the total dose of cyclophosphamide administered to the subject is 100 mg / m². 2 , 125 mg / m² 2 , 150 mg / m² 2 , 175 mg / m² 2 , 200 mg / m² 2 , 225 mg / m² 2 , 250 mg / m² 2 , 275 mg / m² 2 , 300 mg / m² 2 , 350 mg / m² 2 , 375 mg / m² 2 , 400 mg / m² 2 , 450 mg / m² 2 500 mg / m² 2 550 mg / m² 2 , 600 mg / m² 2 , 650 mg / m² 2 700 mg / m² 2 750 mg / m²2 、800 mg / m 2 、850 mg / m 2 、900 mg / m 2 、950 mg / m 2 、1,000 mg / m 2 、1,100 mg / m 2 、1,200 mg / m 2 、1,250 mg / m 2 、1,300 mg / m 2 、1,400 mg / m 2 、1,500 mg / m 2 、1,600 mg / m 2、 1,700 mg / m 2 、1,750 mg / m 2 、1,800 mg / m 2 、1,900 mg / m 2 、 or 2,000 mg / m 2 is. In certain embodiments, the total dose of cyclophosphamide administered to the subject is 500 mg / m 2 is. In certain embodiments, the total dose of cyclophosphamide administered to the subject is 1,000 mg / m 2 is.

[0071] In certain embodiments, the conditioning regimen of the present disclosure consists essentially of administering a dose of cyclophosphamide to the subject on day - 3. In certain embodiments, the dose of cyclophosphamide is 800 - 1,200 mg / m 2 / day (e.g., 800 - 1,200 mg / m 2 / day, 800 - 1,100 mg / m 2 / day, 800 - 1,000 mg / m 2 / day, 800 - 950 mg / m 2 / day, 800 - 900 mg / m 2 / day, 800 - 850 mg / m 2 / day, 850 - 1,200 mg / m 2 / day, 850 - 1,100 mg / m 2 / day, 850 - 1,000 mg / m 2 / day, 850 - 950 mg / m 2 / day, 850 - 900 mg / m 2 / day, 900~1,200mg / m 2 / day, 900~1,100mg / m 2 / day, 900~1,000mg / m 2 / day, 900~950mg / m 2 / day, 950~1,200mg / m 2 / day, 950~1,100mg / m 2 / day, 950~1,000mg / m 2 / day, 1,000~1,200mg / m 2 / day, 1,000~1,100mg / m 2 / day, or 1,100-1,200 mg / m² 2 The dose is 850-1,150 mg / m². In certain embodiments, the dose of cyclophosphamide is 850-1,150 mg / m². 2 For example, 900-1,100 mg / m² per day. 2 For example, 950-1,050 mg / m² per day. 2 The dose is 800 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 800 mg / m². 2 / day, 850mg / m 2 / day, 900mg / m 2 / day, 950mg / m 2 / day, 1,000mg / m 2 / day, 1,100mg / m 2 / day, or 1,200 mg / m² 2 The dose is 1,000 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 1,000 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 800-1,200 mg / m². 2 (For example, 800-1,200 mg / m²) 2 800-1,100 mg / m² 2 800-1,000 mg / m² 2 800-950 mg / m² 2 800-900 mg / m² 2 800-850 mg / m² 2 850-1,200 mg / m² 2 850-1,100 mg / m² 2 850-1,000 mg / m² 2, 850~950 mg / m² 2 850-900 mg / m² 2 900-1,200 mg / m² 2 900-1,100 mg / m² 2 900-1,000 mg / m² 2 900-950 mg / m² 2 950-1,200 mg / m² 2 950-1,100 mg / m² 2 950-1,000 mg / m² 2 1,000-1,200 mg / m² 2 , 1,000~1,100 mg / m² 2 , or 1,100-1,200 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 850-1,150 mg / m². 2 For example, 900-1,100 mg / m² 2 For example, 950-1,050 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 1,000 mg / m². 2 That is the case.

[0072] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering a dose of cyclophosphamide on days -4 and -3, where the dose of cyclophosphamide is 400-600 mg / m². 2 This is / day. For example, in a particular embodiment, the dose of cyclophosphamide is 400-600 mg / m². 2 / day (for example, 400-600 mg / m²) 2 / day, 400~550mg / m 2 / day, 400~500mg / m 2 / day, 400~450mg / m 2 / day, 450~600mg / m 2 / day, 450~550mg / m 2 / day, 450~500mg / m 2 / day, 500~600mg / m 2 / day, 500~550mg / m 2 / day, or 550-600 mg / m² 2 The dose is 450-550 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 450-550 mg / m². 2 The dose is 400 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 400 mg / m². 2 / day, 450mg / m 2 / day, 500mg / m 2 / day, 550mg / m 2 / day, or 600mg / m² 2 The dose is 500 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 500 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 800-1,200 mg / m². 2 (For example, 800-1,200 mg / m²) 2 800-1,100 mg / m² 2 800-1,000 mg / m² 2 800-950 mg / m² 2 800-900 mg / m² 2 800-850 mg / m² 2 850-1,200 mg / m² 2 850-1,100 mg / m² 2 850-1,000 mg / m² 2 , 850~950 mg / m² 2 850-900 mg / m² 2 900-1,200 mg / m² 2 900-1,100 mg / m² 2 900-1,000 mg / m² 2 900-950 mg / m² 2 950-1,200 mg / m² 2 950-1,100 mg / m² 2 950-1,000 mg / m² 2 1,000-1,200 mg / m² 2 , 1,000~1,100 mg / m² 2 , or 1,100-1,200 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 850-1,150 mg / m².2 For example, 900-1,100 mg / m² 2 For example, 950-1,050 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 1,000 mg / m². 2 That is the case.

[0073] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering a dose of cyclophosphamide to target on day 3. In certain embodiments, the dose of cyclophosphamide is 600–900 mg / m² 2 / day (for example, 600-900 mg / m²) 2 / day, 600~850mg / m 2 / day, 600~800mg / m 2 / day, 600~750mg / m 2 / day, 600~700mg / m 2 / day, 600~650mg / m 2 / day, 650~900mg / m 2 / day, 650~850mg / m 2 / day, 650~800mg / m 2 / day, 650~750mg / m 2 / day, 650~700mg / m 2 / day, 700~900mg / m 2 / day, 700~850mg / m 2 / day, 700~800mg / m 2 / day, 700~750mg / m 2 / day, 750~900mg / m 2 / day, 750~850mg / m 2 / day, 750~800mg / m 2 / day, 800~900mg / m 2 / day, 800~850mg / m 2 / day, or 850-900 mg / m² 2 The dose is 650-850 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 650-850 mg / m². 2 / day, for example, 700-800 mg / m² 2 The dose is 600 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 600 mg / m².2 / day, 650mg / m 2 / day, 700mg / m 2 / day, 750mg / m 2 , 800 mg / m² 2 / day, 850mg / m 2 / day, or 900mg / m² 2 The dose is 750 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 750 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 600-900 mg / m². 2 (For example, 600-900 mg / m²) 2 600-850 mg / m² 2 600-800 mg / m² 2 600-750 mg / m² 2 600-700 mg / m² 2 600-650 mg / m² 2 650-900 mg / m² 2 650-850 mg / m² 2 650-800 mg / m² 2 , 650~750 mg / m² 2 , 650-700 mg / m² 2 700-900 mg / m² 2 700-850 mg / m² 2 700-800 mg / m² 2 700-750 mg / m² 2 750-900 mg / m² 2 750-850 mg / m² 2 750-800 mg / m² 2 800-900 mg / m² 2 800-850 mg / m² 2 , or 850-900 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 650-850 mg / m². 2 For example, 700-800 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 750 mg / m². 2 That is the case.

[0074] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering doses of cyclophosphamide to target patients on days -4 and -3. In certain embodiments, the dose of cyclophosphamide is 250–550 mg / m² 2 / day (for example, 250-550 mg / m²) 2 / day, 250~500mg / m 2 / day, 250~450mg / m 2 / day, 250~400mg / m 2 / day, 250~375mg / m 2 / day, 250~350mg / m 2 / day, 250~300mg / m 2 / day, 300~550mg / m 2 / day, 300~500mg / m 2 / day, 300~450mg / m 2 / day, 300~400mg / m 2 / day, 300~375mg / m 2 / day, 300~350mg / m 2 / day, 350~550mg / m 2 / day, 350~500mg / m 2 / day, 350~450mg / m 2 / day, 350~400mg / m 2 / day, 350~375mg / m 2 / day, 375~550mg / m 2 / day, 375~500mg / m 2 / day, 375~450mg / m 2 / day, 375~400mg / m 2 / day, 400~550mg / m 2 / day, 400~500mg / m 2 / day, 400~450mg / m 2 / day, 450~550mg / m 2 / day, 450~500mg / m 2 / day, or 500-550 mg / m² 2 The dose is 300-500 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 300-500 mg / m². 2 For example, 350-400 mg / m² per day. 2The dose is 250 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 250 mg / m². 2 / day, 300mg / m 2 / day, 350mg / m 2 / day, 375mg / m 2 , 400 mg / m² 2 / day, 450mg / m 2 / day, 500mg / m 2 / day, or 550mg / m² 2 The dose is / day. In a particular embodiment, the dose of cyclophosphamide is 375 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 600-900 mg / m². 2 (For example, 600-900 mg / m²) 2 600-850 mg / m² 2 600-800 mg / m² 2 600-750 mg / m² 2 600-700 mg / m² 2 600-650 mg / m² 2 650-900 mg / m² 2 650-850 mg / m² 2 650-800 mg / m² 2 , 650~750 mg / m² 2 , 650-700 mg / m² 2 700-900 mg / m² 2 700-850 mg / m² 2 700-800 mg / m² 2 700-750 mg / m² 2 750-900 mg / m² 2 750-850 mg / m² 2 750-800 mg / m² 2 800-900 mg / m² 2 800-850 mg / m² 2 , or 850-900 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 650-850 mg / m². 2 For example, 700-800 mg / m² 2In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 750 mg / m². 2 That is the case.

[0075] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering doses of cyclophosphamide to target on days -5, -4, and -3. In certain embodiments, the dose of cyclophosphamide is 150-350 mg / m². 2 / day (for example, 150-350 mg / m²) 2 / day, 150~300mg / m 2 / day, 150~250mg / m 2 / day, 150~200mg / m 2 / day, 200~350mg / m 2 / day, 200~300mg / m 2 / day, 200~250mg / m 2 / day, 250~350mg / m 2 / day, 250~300mg / m 2 / day, or 300-350 mg / m² 2 The dose is 200-300 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 200-300 mg / m². 2 For example, 225-275 mg / m² per day. 2 The dose is 150 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 150 mg / m². 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, 300mg / m 2 / day, or 350mg / m² 2 The dose is 250 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 250 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 600-900 mg / m². 2 (For example, 600-900 mg / m²) 2 600-850 mg / m² 2600-800 mg / m² 2 600-750 mg / m² 2 600-700 mg / m² 2 600-650 mg / m² 2 650-900 mg / m² 2 650-850 mg / m² 2 650-800 mg / m² 2 , 650~750 mg / m² 2 , 650-700 mg / m² 2 700-900 mg / m² 2 700-850 mg / m² 2 700-800 mg / m² 2 700-750 mg / m² 2 750-900 mg / m² 2 750-850 mg / m² 2 750-800 mg / m² 2 800-900 mg / m² 2 800-850 mg / m² 2 , or 850-900 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 650-850 mg / m². 2 For example, 700-800 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 750 mg / m². 2 That is the case.

[0076] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering a dose of cyclophosphamide to target on day 3. In certain embodiments, the dose of cyclophosphamide is 400-600 mg / m². 2 / day (for example, 400-600 mg / m²) 2 / day, 400~550mg / m 2 / day, 400~500mg / m 2 / day, 400~450mg / m 2 / day, 450~600mg / m 2 / day, 450~550mg / m 2 / day, 450~500mg / m 2 / day, 500~600mg / m2 / day, 500~550mg / m 2 / day, or 550-600 mg / m² 2 The dose is 450-550 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 450-550 mg / m². 2 The dose is 400 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 400 mg / m². 2 / day, 450mg / m 2 / day, 500mg / m 2 / day, 550mg / m 2 / day, or 600mg / m² 2 The dose is 500 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 500 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 400-600 mg / m². 2 (For example, 400-600 mg / m²) 2 400-550 mg / m² 2 400-500 mg / m² 2 400-450 mg / m² 2 , 450-600 mg / m² 2 , 450~550 mg / m² 2 , 450-500 mg / m² 2 500-600 mg / m² 2 500-550 mg / m² 2 , or 550-600 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 450-550 mg / m². 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 500 mg / m². 2 That is the case.

[0077] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering doses of cyclophosphamide to target patients on days -4 and -3. In certain embodiments, the dose of cyclophosphamide is 150-350 mg / m². 2 / day (for example, 150-350 mg / m²) 2 / day, 150~300mg / m2 / day, 150~250mg / m 2 / day, 150~200mg / m 2 / day, 200~350mg / m 2 / day, 200~300mg / m 2 / day, 200~250mg / m 2 / day, 250~350mg / m 2 / day, 250~300mg / m 2 / day, or 300-350 mg / m² 2 The dose is 200-300 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 200-300 mg / m². 2 For example, 225-275 mg / m² per day. 2 The dose is 150 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 150 mg / m². 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, 300mg / m 2 / day, or 350mg / m² 2 The dose is 250 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 250 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 400-600 mg / m². 2 (For example, 400-600 mg / m²) 2 400-550 mg / m² 2 400-500 mg / m² 2 400-450 mg / m² 2 , 450-600 mg / m² 2 , 450~550 mg / m² 2 , 450-500 mg / m² 2 500-600 mg / m² 2 500-550 mg / m² 2 , or 550-600 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 450-550 mg / m². 2In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 500 mg / m². 2 That is the case.

[0078] In certain embodiments, the preconditioning regimen of the present disclosure essentially consists of administering a dose of cyclophosphamide to the target on day 3. In certain embodiments, the dose of cyclophosphamide is 150–350 mg / m² 2 / day (for example, 150-350 mg / m²) 2 / day, 150~300mg / m 2 / day, 150~250mg / m 2 / day, 150~200mg / m 2 / day, 200~350mg / m 2 / day, 200~300mg / m 2 / day, 200~250mg / m 2 / day, 250~350mg / m 2 / day, 250~300mg / m 2 / day, or 300-350 mg / m² 2 The dose is 200-300 mg / m² / day. In certain embodiments, the dose of cyclophosphamide is 200-300 mg / m². 2 For example, 225-275 mg / m² per day. 2 The dose is 150 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 150 mg / m². 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, 300mg / m 2 / day, or 350mg / m² 2 The dose is 250 mg / m² / day. In a particular embodiment, the dose of cyclophosphamide is 250 mg / m². 2 This is / day. In a particular embodiment, the total dose of cyclophosphamide administered to the subject during the preconditioning regimen is 150-350 mg / m². 2 (For example, 150-350 mg / m²) 2 , 150-300 mg / m² 2, 150-250 mg / m² 2 , 150-200 mg / m² 2 , 200-350 mg / m² 2 , 200-300 mg / m² 2 , 200-250 mg / m² 2 , 250~350 mg / m² 2 , 250-300 mg / m² 2 , or 300-350 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 200-300 mg / m². 2 For example, 225-275 mg / m² 2 In a particular embodiment, the total dose of cyclophosphamide administered to the subject is 250 mg / m². 2 That is the case.

[0079] Table 1 shows some specific exemplary preconditioning regimens in this disclosure. Except for the rightmost column, the doses in Table 1 are mg / m². 2 The dose is shown as cyclophosphamide per day. The total dose (rightmost column in Table 1) is mg / m². 2 It is shown as cyclophosphamide.

[0080] [Table 1]

[0081] Fludarabine is a purine analog that differs from physiological nucleosides in that its sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is commonly used in its 5-O phosphorylated form (i.e., fludarabine phosphate). As used herein, the term “fludarabine” is understood to encompass both the unphosphorylated and 5-O phosphorylated forms. Fludarabine acts as a purine antagonist antimetabolite. When administered to a subject, fludarabine is known to be dephosphorylated to 2-fluoro-ara-A and then phosphorylated intracellularly by deoxycytidine kinase to 2-fluoro-ara-ATP, an active triphosphate ester. This metabolite then interferes with DNA replication, presumably by inhibiting DNA polymerase and ribonucleotide reductase, thereby inhibiting DNA synthesis (Gandhi and Plunkett (2002) Clin. Pharmacokinet. 41:93-103). As a result, administration of fludarabine leads to increased cell death in dividing cells. Fludarabine is used to treat a variety of diseases or disorders, including hematological malignancies such as lymphoma and leukemia. Fludarabine is also used in conditioning treatments administered to patients before allogeneic stem cell transplantation or adoptive cell therapy. In the context of adoptive T-cell therapy for cancer treatment, the use of fludarabine in lymphocyte depletion preconditioning regimens has been previously shown to improve the efficacy and persistence of T cells and improve event-free survival in cancer patients (Hay et al. (2019) Blood 133(15):1652-1663, Ramachandran et al. (2019) J.Immunother.Cancer 7:276).

[0082] In certain embodiments, the preconditioning regimen of the Disclosure does not involve administering a dose of fludarabine to the subject. In certain embodiments of the treatment of the Disclosure, the subject is not administered fludarabine. In certain embodiments, the subject is not administered fludarabine, but the autoimmune disease is treated by the method of the Disclosure.

[0083] In certain embodiments, the therapeutic method of the present disclosure comprises (a) administering to a subject a preconditioning regimen comprising or essentially comprising one or more doses of cyclophosphamide; and (b) administering to the subject a therapeutically effective amount of genetically engineered immune cells (e.g., CAR immune cells, e.g., CAR T cells) after step (a). In certain embodiments, fludarabine is not administered to the subject within 10 days prior to step (b) (e.g., within 10 days prior to the start of step (b)). In certain embodiments, fludarabine is not administered to the subject within 7 days prior to step (b) (e.g., within 7 days prior to the start of step (b)). In certain embodiments, fludarabine is not administered to the subject within 5 days prior to step (b) (e.g., within 5 days prior to the start of step (b)). In certain embodiments, fludarabine is not administered to the subject on 10, 9, 8, 7, 6, 5, 4, 3, 2, and / or 1 day prior to step (b) (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, and / or 1 day prior to the start of step (b)). In certain embodiments, step (b) is defined as taking place on "Day 0", where "-X Day" and "X Day" refer to the days that are X days before and X days after Day 0, respectively. In certain embodiments, fludarabine is not administered to the subject on or after Day -10 and before Day 0. In certain embodiments, fludarabine is not administered to the subject on or after Day -7 and before Day 0. In certain embodiments, fludarabine is not administered to the subject on or after Day -6 and before Day 0. In certain embodiments, fludarabine is not administered to the subject on or after Day -5 and before Day 0. In certain embodiments, fludarabine is not administered to the subject on day -10, day -9, day -8, day -7, day -6, day -5, day -4, day -3, day -2, day -1, and / or day 0. In certain embodiments, fludarabine is not administered to the subject after day -10, day -9, day -8, day -7, day -6, day -5, day -4, day -3, day -2, day -1, and / or day 0.

[0084] In certain embodiments, the preconditioning regimen of the present disclosure includes administering one or more doses of cyclophosphamide to the target, and if a dose of fludarabine is also administered to the target, the dose of fludarabine is 2 mg / m². 2 Less than 3 mg / m² per day 2 Less than 4 mg / m² per day, 4 mg / m² 2 Less than 5 mg / m² per day, 5 mg / m² 2 Less than 6 mg / m² per day 2 Less than 6.25 mg / m² per day. 2 Less than 6.5 mg / m² per day. 2 Less than 7 mg / m² per day 2 Less than 7.5 mg / m² per day. 2 Less than 8 mg / m² per day 2 Less than 9 mg / m² per day 2 Less than 10 mg / m² per day, 10 mg / m² 2 Less than 11 mg / m² per day, 11 mg / m² 2 Less than 12 mg / m² per day, 12 mg / m² 2 Less than 12.5 mg / m² per day. 2 Less than 13 mg / m² per day, 13 mg / m² 2 Less than 14 mg / m² per day, 14 mg / m² 2 Less than 15 mg / m² per day. 2 Less than 16 mg / m² per day. 2 Less than 17 mg / m² per day. 2 Less than 18 mg / m² per day. 2 Less than 19 mg / m² per day. 2 Less than 20 mg / m² per day. 2 Less than 22.5 mg / m² per day. 2 Less than 25 mg / m² per day, or 25 mg / m². 2 It is less than / day.

[0085] In certain embodiments, the preconditioning regimen of the present disclosure includes administering one or more doses of cyclophosphamide to the subject, and if a dose of fludarabine is also administered to the subject, the total dose of fludarabine administered to the subject is 75 mg / m². 2 Less than 67.5 mg / m² 2 Less than 60 mg / m² 2 Less than 57 mg / m² 2 Less than 54 mg / m² 2 Less than 51 mg / m² 2 Less than 50 mg / m² 2Less than 48 mg / m² 2 Less than 45 mg / m² 2 Less than 42 mg / m² 2 Less than 39 mg / m² 2 Less than 37.5 mg / m² 2 Less than 36 mg / m² 2 Less than 33 mg / m² 2 Less than 30 mg / m² 2 Less than 27 mg / m² 2 Less than 25 mg / m² 2 Less than 24 mg / m² 2 Less than 21 mg / m² 2 Less than 19.5 mg / m² 2 Less than 18.75 mg / m² 2 Less than 18 mg / m² 2 Less than 15 mg / m² 2 Less than 12 mg / m² 2 Less than 9 mg / m² 2 Less than 6 mg / m² 2 It is less than.

[0086] This disclosure further relates to pharmaceutical compositions comprising, or essentially comprising, a dose of cyclophosphamide and a pharmaceutically acceptable carrier, as disclosed herein. In certain embodiments, cyclophosphamide is administered intravenously to a subject.

[0087] b.Cell therapy In certain embodiments, the therapeutic methods described herein include administering an immunotherapy to a target. For example, in certain embodiments, the therapeutic treatment method described herein includes administering an immune cell composition comprising immune cells (e.g., T cells, B cells, NK cells, tumor-infiltrating lymphocytes, and / or dendritic cells) to a target. In certain embodiments, the immune cell composition comprises NK cells. In certain embodiments, the immune cell composition comprises T cells, e.g., CD8+ T cells. In certain embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the immune cell composition are CD8+ T cells. In certain embodiments, the immune cell composition further comprises CD4+ T cells, where optionally, at least 20%, at least 30%, at least 40%, or at least 50% of the cells in the immune cell composition are CD4+ T cells.

[0088] Immune cells used in immunotherapy may be obtained from a patient's blood sample by apheresis. In certain embodiments, lymphocyte-rich and monocyte-rich fractions may be obtained by elutriation of the patient's peripheral blood mononuclear cells (PBMCs). The monocyte-rich fraction may then be used to prepare antigen-presenting cells (APCs) for priming T cells that may be obtained from the lymphocyte-rich fraction. Exemplary methods are described in WO2020055931 and U.S. Patents 9,963,677 and 9,642,906. In certain embodiments, the immunotherapy is autologous, i.e., immune cells obtained from a patient are cultured in vitro and then administered to the same patient. In certain embodiments, the immunotherapy is allogeneic, where the immune cells are optionally genetically engineered to suppress the expression of class I MHC (e.g., β2M), class II (e.g., RFXANK), or TCR (e.g., TRAC or CD3) components by inactivating them.

[0089] In certain embodiments, the immune cells used in cell therapy are T cells, such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, γδ T cells, natural killer cells, cytokine-induced killer cells, their cell lines, memory T stem cells, or other T effector cells. In certain embodiments, the T cells specifically target cells expressing a targeted antigen, such as B cells expressing CD19. In certain embodiments, the T cells are self to the target. In other embodiments, the T cells are allogeneic to the target.

[0090] Immune cells (e.g., T cells, e.g., CD8 + T cells may possess antigen specificity through receptors expressed on their cell surface. T cells may spontaneously express T cell receptors (TCRs), such as αβTCR or γδTCR, and may be "trained" to express TCRs that target a given antigen or epitope. Immune cells may also be genetically engineered to express recombinant TCRs or chimeric antigen receptors (CARs). When therapies disclosed herein are used to treat autoimmune diseases, the TCR or CAR may target one or more antigens or epitopes present on the surface of immune cells. In certain embodiments, immune cells are genetically engineered to inactivate their endogenous TCRs, for example by knocking out the TRAC or TRBC gene, in order to reduce ligand-independent tonic T cell signaling and enhance T cell potency.

[0091] In certain embodiments, immune cells (e.g., T cells or NK cells) used in immunotherapy express CARs as described herein. In certain embodiments, immune cells used in immunotherapy express recombinant TCRs. Generally, αβ TCRs can bind to peptides presented by major histocompatibility complex (MHC) molecules, while γδ TCRs do not require MHC-mediated antigen presentation. In certain embodiments, TCRs include variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively), or portions thereof that bind to an antigen (e.g., peptide-MHC complex) including α and β chain variable domains or γ and δ chain variable domains. Generally, the variable domains of TCRs include complementarity-determining regions involved in the recognition of antigens (e.g., peptide-MHC complexes). In certain embodiments, the TCRs expressed by immune cells are cloned from naturally occurring T cells.

[0092] Immune cells used in immunotherapy can be genetically engineered to express a CAR or TCR by introducing a nucleic acid encoding the CAR or TCR. In certain embodiments, the nucleic acid is a DNA molecule (e.g., a cDNA molecule). In certain embodiments, the nucleic acid further includes an expression control sequence (e.g., a promoter and / or enhancer) operably linked to the sequence encoding the CAR or TCR. In certain embodiments, the immune cells are transduced by a vector containing the nucleic acid encoding the CAR or TCR, e.g., a viral vector (e.g., an AAV vector, a lentiviral vector, or an adenovirus vector) or a non-viral vector (e.g., a plasmid). In certain embodiments, the nucleic acid is an RNA molecule (e.g., an mRNA molecule). Methods for preparing and modifying mRNA used for transduction are disclosed in U.S. Patents 8,278,036, 8,883,506, and 8,716,465. In certain embodiments, the nucleic acid encodes an amino acid sequence containing a signal peptide at the N-terminus of the CAR or TCR. Such signal peptides may promote the cell surface localization of CARs or TCRs when they are expressed in effector cells and cleaved from CARs during intracellular processing.

[0093] In certain embodiments, the therapeutic methods of the present disclosure include administering an effective amount of immune cells (e.g., CAR immune cells, e.g., CAR T cells) to a subject in need. In certain embodiments, the effective amount of immune cells is 1 × 10⁻⁶ 5 Individual cells / kg body weight~1×10 8 This is expressed as individual cells / kg body weight. For example, in a particular embodiment, the effective amount of immune cells is 1 × 10⁻⁶ 5 ~1 × 10 8 Individual cells / kg, 1×10 5 ~9×10 7 Individual cells / kg, 1×10 5 ~6×10 7 Individual cells / kg, 1×10 5 ~3×10 7 Individual cells / kg, 1×10 5 ~1 × 10 7cells / kg, 1×10 5 ~9×10 6 cells / kg, 1×10 5 ~8×10 6 cells / kg, 1×10 5 ~7×10 6 cells / kg, 1×10 5 ~6×10 6 cells / kg, 1×10 5 ~5×10 6 cells / kg, 1×10 5 ~4×10 6 cells / kg, 1×10 5 ~3×10 6 cells / kg, 1×10 5 ~2×10 6 cells / kg, 1×10 5 ~1×10 6 cells / kg, 1×10 5 ~9×10 5 cells / kg, 1×10 5 ~6×10 5 cells / kg, 1×10 5 ~3×10 5 Cells / kg, 3×10 5 ~1×10 8 Cells / kg, 3×10 5 ~9×10 7 Cells / kg, 3×10 5 ~6×10 7 Cells / kg, 3×10 5 ~3×10 7 Cells / kg, 3×10 5 ~1×10 7 Cells / kg, 3×10 5 ~9×10 6 Cells / kg, 3×10 5 ~8×10 6 Cells / kg, 3×10 5 ~7×10 6 Cells / kg, 3×10 5 ~6×10 6 Cells / kg, 3×10 5 ~5×10 6 Cells / kg, 3×10 5 ~4×10 6 Cells / kg, 3×10 5 ~3×106 Cells / kg, 3×10 5 ~2×10 6 Cells / kg, 3×10 5 ~1×10 6 Cells / kg, 3×10 5 ~9×10 5 Cells / kg, 3×10 5 ~6×10 5 Cells / kg, 6×10 5 ~1×10 8 Cells / kg, 6×10 5 ~9×10 7 Cells / kg, 6×10 5 ~6×10 7 Cells / kg, 6×10 5 ~3×10 7 Cells / kg, 6×10 5 ~1×10 7 Cells / kg, 6×10 5 ~9×10 6 Cells / kg, 6×10 5 ~8×10 6 Cells / kg, 6×10 5 ~7×10 6 Cells / kg, 6×10 5 ~6×10 6 Cells / kg, 6×10 5 ~5×10 6 Cells / kg, 6×10 5 ~4×10 6 Cells / kg, 6×10 5 ~3×10 6 Cells / kg, 6×10 5 ~2×10 6 Cells / kg, 6×10 5 ~1×10 6 Cells / kg, 6×10 5 ~9×10 5 Cells / kg, 9×10 5 ~1×10 8 Cells / kg, 9×10 5 ~9×10 7 Cells / kg, 9×10 5 ~6×10 7 Cells / kg, 9×10 5 ~3×10 7 Cells / kg, 9×10 5~1×10 7 Cells / kg, 9×10 5 ~9×10 6 Cells / kg, 9×10 5 ~8×10 6 Cells / kg, 9×10 5 ~7×10 6 Cells / kg, 9×10 5 ~6×10 6 Cells / kg, 9×10 5 ~5×10 6 Cells / kg, 9×10 5 ~4×10 6 Cells / kg, 9×10 5 ~3×10 6 Cells / kg, 9×10 5 ~2×10 6 Cells / kg, 9×10 5 ~1×10 6 cells / kg, 1×10 6 ~1×10 8 cells / kg, 1×10 6 ~9×10 7 cells / kg, 1×10 6 ~6×10 7 cells / kg, 1×10 6 ~3×10 7 cells / kg, 1×10 6 ~1×10 7 cells / kg, 1×10 6 ~9×10 6 cells / kg, 1×10 6 ~8×10 6 cells / kg, 1×10 6 ~7×10 6 cells / kg, 1×10 6 ~6×10 6 cells / kg, 1×10 6 ~5×10 6 cells / kg, 1×10 6 ~4×10 6 cells / kg, 1×10 6 ~3×10 6 cells / kg, 1×10 6 ~2×10 6 cells / kg, 2×10 6 ~1×10 8 cells / kg, 2×106 ~9×10 7 cells / kg, 2×10 6 ~6×10 7 cells / kg, 2×10 6 ~3×10 7 cells / kg, 2×10 6 ~1×10 7 cells / kg, 2×10 6 ~9×10 6 cells / kg, 2×10 6 ~8×10 6 cells / kg, 2×10 6 ~7×10 6 cells / kg, 2×10 6 ~6×10 6 cells / kg, 2×10 6 ~5×10 6 cells / kg, 2×10 6 ~4×10 6 cells / kg, 2×10 6 ~3×10 6 Cells / kg, 3×10 6 ~1×10 8 Cells / kg, 3×10 6 ~9×10 7 Cells / kg, 3×10 6 ~6×10 7 Cells / kg, 3×10 6 ~3×10 7 Cells / kg, 3×10 6 ~1×10 7 Cells / kg, 3×10 6 ~9×10 6 Cells / kg, 3×10 6 ~8×10 6 Cells / kg, 3×10 6 ~7×10 6 Cells / kg, 3×10 6 ~6×10 6 Cells / kg, 3×10 6 ~5×10 6 Cells / kg, 3×10 6 ~4×10 6 cells / kg, 4×10 6 ~1×10 8 cells / kg, 4×10 6 ~9×10 7cells / kg, 4×10 6 ~6×10 7 cells / kg, 4×10 6 ~3×10 7 cells / kg, 4×10 6 ~1×10 7 cells / kg, 4×10 6 ~9×10 6 cells / kg, 4×10 6 ~8×10 6 cells / kg, 4×10 6 ~7×10 6 cells / kg, 4×10 6 ~6×10 6 cells / kg, 4×10 6 ~5×10 6 cells / kg, 5×10 6 ~1×10 8 cells / kg, 5×10 6 ~9×10 7 cells / kg, 5×10 6 ~6×10 7 cells / kg, 5×10 6 ~3×10 7 cells / kg, 5×10 6 ~1×10 7 cells / kg, 5×10 6 ~9×10 6 cells / kg, 5×10 6 ~8×10 6 cells / kg, 5×10 6 ~7×10 6 cells / kg, 5×10 6 ~6×10 6 Cells / kg, 6×10 6 ~1×10 8 Cells / kg, 6×10 6 ~9×10 7 Cells / kg, 6×10 6 ~6×10 7 Cells / kg, 6×10 6 ~3×10 7 Cells / kg, 6×10 6 ~1×10 7 Cells / kg, 6×10 6 ~9×10 6 Cells / kg, 6×10 6 ~8×106 Cells / kg, 6×10 6 ~7×10 6 Cells / kg, 7×10 6 ~1×10 8 Cells / kg, 7×10 6 ~9×10 7 Cells / kg, 7×10 6 ~6×10 7 Cells / kg, 7×10 6 ~3×10 7 Cells / kg, 7×10 6 ~1×10 7 Cells / kg, 7×10 6 ~9×10 6 Cells / kg, 7×10 6 ~8×10 6 Cells / kg, 8×10 6 ~1×10 8 Cells / kg, 8×10 6 ~9×10 7 Cells / kg, 8×10 6 ~6×10 7 Cells / kg, 8×10 6 ~3×10 7 Cells / kg, 8×10 6 ~1×10 7 Cells / kg, 8×10 6 ~9×10 6 Cells / kg, 9×10 6 ~1×10 8 Cells / kg, 9×10 6 ~9×10 7 Cells / kg, 9×10 6 ~6×10 7 Cells / kg, 9×10 6 ~3×10 7 Cells / kg, 9×10 6 ~1×10 7 cells / kg, 1×10 7 ~1×10 8 cells / kg, 1×10 7 ~9×10 7 cells / kg, 1×10 7 ~6×10 7 cells / kg, 1×10 7 ~3×10 7 Cells / kg, 3×10 7~1 × 10 8 Individual cells / kg, 3×10 7 ~9×10 7 Individual cells / kg, 3×10 7 ~6×10 7 Individual cells / kg, 6×10 7 ~1 × 10 8 Individual cells / kg, 6×10 7 ~9×10 7 Individual cells / kg, or 9 × 10⁶ 7 ~1 × 10 8 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is 1 × 10⁻⁶ 6 ~1 × 10 7 It is expressed as individual cells / kg.

[0094] In a particular embodiment, the effective amount of immune cells is at least 1 × 10⁻⁶ 5 This is expressed as individual cells / kg. For example, in a particular embodiment, the effective amount of immune cells is at least 1 × 10⁶ 8 Individual cells / kg, at least 9 × 10⁶ 7 Individual cells / kg, at least 6 × 10⁶ 7 Individual cells / kg, at least 3 × 10⁶ 7 Individual cells / kg, at least 1 × 10⁶ 7 Individual cells / kg, at least 9 × 10⁶ 6 Individual cells / kg, at least 8 × 10⁶ 6 Individual cells / kg, at least 7 × 10⁶ 6 Individual cells / kg, at least 6 × 10⁶ 6 Individual cells / kg, at least 5 × 10⁶ 6 Individual cells / kg, at least 4 × 10⁶ 6 Individual cells / kg, at least 3 × 10⁶ 6 Individual cells / kg, at least 2 × 10⁶ 6 Individual cells / kg, at least 1 × 10⁶ 6 Individual cells / kg, at least 9 × 10⁶ 5 Individual cells / kg, at least 6 × 10⁶ 5 Individual cells / kg, at least 3 × 10⁶ 5 Individual cells / kg, or at least 1 × 10⁶ 5 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is at least 3 × 10⁶ 5Individual cells / kg, at least 1 × 10⁶ 6 Individual cells / kg, at least 3 × 10⁶ 6 Individual cells / kg, or at least 1 × 10⁶ 7 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is at least 1 × 10⁶ 6 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is 1 × 10⁻⁶ 8 Individual cells / kg, 9×10 7 Individual cells / kg, 6×10 7 Individual cells / kg, 3×10 7 Individual cells / kg, 1×10 7 Individual cells / kg, 9×10 6 individual cells / kg, 8×10 6 Individual cells / kg, 7×10 6 Individual cells / kg, 6×10 6 Individual cells / kg, 5×10 6 Individual cells / kg, 4×10 6 Individual cells / kg, 3×10 6 Individual cells / kg, 2×10 6 Individual cells / kg, 1×10 6 Individual cells / kg, 9×10 5 Individual cells / kg, 6×10 5 Individual cells / kg, 3×10 5 Individual cells / kg, or 1 × 10⁶ 5 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is 3 × 10⁻⁶ 5 Individual cells / kg, 1×10 6 Individual cells / kg, 3×10 6 Individual cells / kg, or 1 × 10⁶ 7 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is 1 × 10⁻⁶ 6 The value is individual cells / kg. In a particular embodiment, the effective amount of immune cells is 1 × 10⁻⁶ 7 It is expressed as individual cells / kg.

[0095] In certain embodiments, an effective dose of immune cells is administered to the subject in a single dose. In certain embodiments, an effective dose of immune cells is administered to the subject at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 days after completion of the preconditioning regimen. In certain embodiments, an effective dose of immune cells is administered intravenously.

[0096] This disclosure further relates to a pharmaceutical composition comprising an effective amount of immune cells as described herein (e.g., T cells, e.g., CAR T cells). In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0097] c. Therapeutic use This disclosure provides methods for reducing an undesirable immune response in a subject by (1) administering a preconditioning regimen as described herein (e.g., preconditioning regimens summarized in Table 1) to the subject, and (2) administering an effective amount of genetically modified immune cells (e.g., CAR immune cells, e.g., CAR T cells), where the immune cells target and kill target immune cells (e.g., B cells), thereby reducing the undesirable immune response in the subject. This disclosure also provides methods for reducing an undesirable immune response in a subject by administering an effective amount of CAR T cells containing nucleic acids encoding CD19-specific CARs, e.g., CARs as described in Section III below, e.g., CARs having CDRs corresponding to the "CD19-1" binders shown in Tables 2-4.

[0098] In certain cases, an undesirable immune response is mediated, at least partially, by the activity of immunoglobulins. Immunoglobulins are glycoproteins belonging to the immunoglobulin superfamily that recognize antigens and facilitate the humoral response of the immune system. Immunoglobulins can exist in two physical forms: a soluble form secreted from cells and a membrane-bound form called the B cell receptor (BCR), which is bound to the surface of B cells. Immature B cells that have not been exposed to an antigen are known as naive B cells and express only the cell surface-bound IgM isotype. Once B cells reach maturity, they begin to express both IgM and IgD, indicating that they are ready to react to an antigen. B cell activation occurs after the binding of the BCR to the antigen, causing the cell to divide and differentiate into antibody-producing plasma cells. In this activated form, B cells begin to produce secretory antibodies rather than membrane-bound antibodies. However, B cells that produce antibodies directed against self-antigens (autoantibodies) can lead to undesirable immune responses against the organism's own cells and tissues, thereby contributing to autoimmune disorders.

[0099] Accordingly, this disclosure provides a method for treating an autoimmune disease of interest. The method comprises administering a preconditioning regimen described herein (e.g., the preconditioning regimens shown in Table 1) to the subject, and administering an effective amount of genetically engineered immune cells (e.g., CAR immune cells, e.g., CAR T cells) that target immune cells, e.g., B cells. In certain embodiments, the method is used to reduce the number of circulating B cells in the subject. In certain embodiments, the B cells in the subject produce autoantibodies that have specificity for autoantigens, e.g., anti-dsDNA antibodies. In certain embodiments, the method is used to reduce the number of antibodies, e.g., autoantibodies, in the subject. In certain embodiments, the autoimmune disease is a B-cell mediated autoimmune disease, i.e., an autoimmune disease where (1) at least some symptoms or signs of the disease are caused by and / or exacerbated by immunoglobulins produced by the B cells of the subject, and / or (2) the B cells play some role in the development or maintenance of the disease or its symptoms or signs.

[0100] Examples of B-cell-mediated autoimmune diseases, though not limited to these, include: systemic lupus erythematosus (SLE), lupus nephritis, SLE with anti-dsDNA antibodies, pemphigus vulgaris (PV), mucosal PV, mucocutaneous PV, myasthenia gravis (MG), MuSK-associated MG, and AChR. Myositis, myositis, membranous nephropathy, antisynthetic enzyme syndrome, dermatomyositis, and immune-mediated necrotizing myopathy, multiple sclerosis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, anti-NMDA receptor encephalitis, Lambert-Eaton syndrome, pemphigus foliaceus, acquired epidermolysis bullosa, bullous pemphigoid, Goodpasture syndrome, rheumatoid arthritis, systemic sclerosis, anti-human neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, immune thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, type 1 diabetes mellitus, Graves' disease, Hashimoto's disease, and Sjögren's syndrome. In certain embodiments, the treatments described herein are used to treat any of the aforementioned autoimmune diseases in a subject. In certain embodiments, the therapies described herein are used to treat SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK-related MG, AChR MG, myositis, membranous nephropathy, anti-synthase syndrome, dermatomyositis, or immune-mediated necrotizing myopathy in subjects who require treatment for SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK-related MG, AChR MG, myositis, membranous nephropathy, anti-synthase syndrome, dermatomyositis, or immune-mediated necrotizing myopathy. In certain embodiments, the therapies described herein are used to treat SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK-related MG, AChR MG, myositis, membranous nephropathy, anti-synthase syndrome, dermatomyositis, or immune-mediated necrotizing myopathy in subjects who require treatment for SLE, lupus nephritis, SLE with anti-dsDNA antibodies, PV, mucosal PV, mucocutaneous PV, MG, MuSK-related MG, AChR MG, myositis, membranous nephropathy, anti-synthase syndrome, dermatomyositis, or immune-mediated necrotizing myopathy.

[0101] In certain embodiments, treatment of a subject using the method of the Disclosure results in a reduction in the autoantibody level in the subject (e.g., anti-dsDNA antibody level or antinuclear antibody level). For example, in certain embodiments, the autoantibody level in the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% compared to the autoantibody level in the subject before treatment. In certain embodiments, treatment of a subject by the method of the Disclosure makes the autoantibody level in the subject undetectable. Autoantibody levels can be measured using any suitable technique known in the art, for example, by ELISA.

[0102] In certain embodiments, treatment of a subject using the method of the Disclosure results in a reduction in circulating B cell levels. For example, in certain embodiments, the B cell level in a subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% compared to the circulating B cell level in the subject before treatment. In certain embodiments, treatment of a subject by the method of the Disclosure makes the circulating B cell level in the subject undetectable. In certain embodiments, the decrease in circulating B cells is transient, and therefore, B cell levels partially or completely recover after a certain period of time.

[0103] In certain embodiments, treatment of a subject using the method of the Disclosure results in a reduction in the amount of circulating CD19+ cells. For example, in certain embodiments, the amount of circulating CD19+ cells in a subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% compared to the level of circulating B cells in the subject before treatment. In certain embodiments, treatment of a subject by the method of the Disclosure makes the amount of circulating CD19+ cells in the subject undetectable. In certain embodiments, the decrease in the amount of circulating CD19+ cells is transient, and levels partially or completely recover after a certain period of time.

[0104] In certain embodiments, the therapeutic methods of this disclosure may be used to treat a target SLE.

[0105] In certain embodiments, treatment of a subject with lupus nephritis using the method of the Disclosure results in a reduction of proteinuria, as measured, for example, by urinary protein concentration. For example, in certain embodiments, the proteinuria of the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, compared, for example, with respect to the subject before treatment or compared to a suitable control subject or control group that did not receive treatment. In certain embodiments, treatment of a subject by the method of the Disclosure results in the elimination of proteinuria.

[0106] In certain embodiments, treatment of a target SLE using the method of the present disclosure results in an enhancement of the amount or activity of one or more complement factors, as can be detected, for example, by measuring serum levels of complement component 3 (C3) or complement component 4 (C4), or by measuring 50% hemolytic complement (CH50) activity. For example, in a particular embodiment, the amount of complement factor in a subject increases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1,000% compared to, for example, the amount of complement factor in a subject before treatment, or compared to a suitable control subject or control group that did not receive treatment. In a particular embodiment, the CH50 activity in a subject is enhanced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to, for example, the CH50 activity in the subject before treatment, or compared to a suitable control subject or control group that did not receive treatment.

[0107] In certain embodiments, treatment of a subject's autoimmune disease using the method of the present disclosure results in a reduction of disease symptoms or disease activity. For example, in certain embodiments, treatment of a subject's SLE (e.g., lupus nephritis) using the method of the present disclosure results in a reduction of disease activity, as measured by an SLE disease activity index or scale, such as the SLEDAI-2K Responder Index, the Commonwealth Lupus Assessment Committee (BILAG) Index, the Physician's Global Assessment Scale, the Skin LE Area and Severity Index (CLASI), the SLICC Disorder Index (SDI), and / or the counting of tender and swollen joints. In certain embodiments, after treatment of the subject's SLE, the subject meets the criteria for complete renal remission (CRR), SRI-4 responder criteria, SRI-5 responder criteria, SRI-6 responder criteria, BICLA responder criteria, LLDAS criteria, and / or DORIS remission criteria.

[0108] In certain embodiments in which the treatment of the present disclosure is used to treat SLE in subjects requiring treatment for SLE, the subject's SLEDAI-2K score is reduced. For example, in certain embodiments, the subject's SLEDAI-2K score is reduced by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, or at least 30 compared to, for example, the subject's score before treatment, or compared to a suitable control subject or control group that did not receive treatment. In certain embodiments, the subject's SLEDAI-2K score is reduced to 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2, or 0-1. In certain embodiments, the subject's SLEDAI-2K score is reduced to 5, 4, 3, 2, 1, or 0. In certain embodiments, the subject's SLEDAI-2K score is reduced to 0.

[0109] In certain embodiments in which the treatment of the present disclosure is used to treat SLE in subjects requiring treatment for SLE, the subject's BILAG-2004 score decreases. For example, in certain embodiments, the subject's BILAG score decreases by at least one grade, at least two grades, or three grades compared to, for example, the subject's BILAG score before treatment by the method of the present disclosure, or compared to a suitable control subject or control group that did not receive treatment. In certain embodiments, the subject's BILAG score decreases by one, two, or three grades. In certain embodiments, the subject's BILAG score decreases to grade B, grade C, or grade D.

[0110] In certain embodiments in which the treatment of the present disclosure is used to treat SLE in subjects who require treatment for SLE, the subject's PGA score decreases after treatment by the method of the present disclosure. For example, in certain embodiments, the subject's PGA score decreases by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.8, at least 1, at least 1.5, at least 2, or at least 2.5 compared to, for example, the subject's PGA score before treatment by the method of the present disclosure, or compared to a suitable control subject or control group that did not receive treatment.

[0111] In certain embodiments in which the treatment of the present disclosure is used to treat SLE in subjects who require treatment for SLE, the subject's CLASI score decreases after treatment by the method of the present disclosure. For example, in certain embodiments, the subject's CLASI score decreases by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, or at least 30 compared to, for example, the subject's CLASI score before treatment by the method of the present disclosure, or compared to a suitable control subject or control group that did not receive treatment.

[0112] In certain embodiments, the treatments of the present disclosure may be used to treat a target myositis (idiopathic inflammatory muscle disease, e.g., dermatomyositis (DM), antisynthesis syndrome (ASyS), and immune-mediated necrotizing myopathy (IMNM)). In certain embodiments, treatment of a target myositis using the methods of the present disclosure results in a reduction of disease symptoms or activity, as measured by an appropriate indicator, assessment, or scale, e.g., one of the following: MMT-8, Patient Global Assessment of Disease Activity (PGA), Physician Global Assessment of Disease Activity (MDGA), Health Assessment Questionnaire Disability Index (HAQ-DI), Extramuscular Global Assessment of Disease Activity (Myositis Disease Activity Assessment Tool [MDAAT]), and Numerical Rating Scale for Pain and Fatigue (NRS).

[0113] In certain embodiments, treatment of a subject with myositis using the method of the present disclosure results in a reduction in serum levels of enzymes in the subject, such as creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), aldolase, myoglobin, or lactate dehydrogenase (LDH). For example, in certain embodiments, the serum level of the enzyme in the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared, for example, the serum level of the enzyme in the subject before treatment, or compared to a suitable control subject or control group that did not receive treatment.

[0114] In certain embodiments, treatment of a subject using the method of the Disclosure results in a reduction in the level of myositis-specific antibodies (MSA) in the subject. For example, in certain embodiments, the MSA level in the subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% compared to the MSA level in the subject before treatment. In certain embodiments, treatment of a subject by the method of the Disclosure makes the MSA level in the subject undetectable.

[0115] In certain embodiments, the treatment methods of the present disclosure may be used to treat pemphigus vulgaris in a subject. In certain embodiments, treatment of pemphigus vulgaris in a subject using the methods of the present disclosure results in a reduction of disease symptoms or disease activity, as measured by an appropriate index, assessment, or scale, such as the Pemphigus Disease Activity Index (PDAI). For example, in certain embodiments, the subject's PDAI score is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared, for example, the subject's score before treatment or compared to an appropriate control subject or control group that did not receive treatment.

[0116] In certain embodiments, the treatments of the present disclosure may be used to treat systemic sclerosis in a subject. In certain embodiments, treatment of systemic sclerosis in a subject using the methods of the present disclosure results in a reduction of disease symptoms or activity, as measured by an appropriate index, assessment, or scale, for example, the following: the Combined Response Index of Systemic Sclerosis (CRISS), Forced Vital Capacity (FVC), Modified Rodnan Skin Score (mRSS), Disability Index on Health Assessment Questionnaire (HAQ-DI), Patient Global Assessment (PtGA), Physician Global Assessment (PGA), Scleroderma Health Assessment Questionnaire (SHAQ), Scleroderma Skin Patient Reported Outcomes (SSPRO), University of California, Los Angeles Scleroderma Clinical Trials Consortium Gastrointestinal Scale (UCLA SCTC GIT), Functional Assessment of Chronic Disease Treatment - Fatigue (FACIT-F), Numerical Rating Scale of Pain (NRS), 36-Item Short Health Questionnaire (SF-36), and EuroQol 5-Item 5-Level Questionnaire (EQ-5D-5L).

[0117] In certain embodiments, the treatment methods of the present disclosure may be used to treat a subject with myasthenia gravis. In certain embodiments, treatment of a subject with myasthenia gravis using the methods of the present disclosure results in a reduction of disease symptoms or activity, as measured by an appropriate index, assessment, or scale, for example, one of the following: Myasthenia Gravis-Activities of Daily Living (MG-ADL), Quantitative Myasthenia Gravis Score (QMG), Myasthenia Gravis Composite Scale (MGC), Post-Intervention Status of the Myasthenia Gravis Foundation of America (MGFA-PIS), MG Disability Index (MGII), 15-Item Myasthenia Gravis Quality of Life Scale (MG-QOL 15r), Quality of Life in Neurological Disorders Scale (Neuro-QoL), and EQ-5D-5L.

[0118] In certain embodiments, treatment of a subject having myasthenia gravis using the method of the present disclosure results in a reduction in autoantibody levels in the subject (e.g., anti-acetylcholine receptor (anti-AChR) autoantibody, anti-muscle-specific kinase (anti-MuSK) autoantibody, and / or anti-low-density lipoprotein receptor-related protein 4 (anti-LRP4) autoantibody). For example, in certain embodiments, the levels of anti-AChR autoantibodies, anti-MuSK autoantibodies, and / or anti-LRP4 autoantibodies in a subject are reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% compared to the autoantibody levels in the subject before treatment. In certain embodiments, treatment of a subject by the method of the present disclosure makes the autoantibody levels in the subject undetectable. Autoantibody levels can be measured using any suitable technique known in the art, for example, by ELISA.

[0119] The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or therapeutic modalities. As used herein, the term “administered in combination” is understood to mean that two (or more) different treatments are delivered to a subject so that the effects of the treatments on the patient overlap at a given point in time during the course of the subject’s illness. In certain other embodiments, the delivery of one treatment is still taking place when the delivery of the second treatment begins, and as a result, there is an overlap in administration. This may be referred to herein as “simultaneous delivery” or “concurrent delivery.” In certain embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. In certain embodiments of either case, the treatments are more effective due to the combination of administrations. For example, the second treatment is more effective, for instance, an equivalent effect may be observed with fewer second treatments, or the second treatment reduces symptoms to a greater extent than would be observed if the second treatment were administered without the first treatment, or a similar situation may be observed with the first treatment. In some embodiments, the delivery is such that the reduction of other parameters related to the symptom or disorder is greater than the reduction observed when one treatment is delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the delivered first treatment is still detectable when the second treatment is delivered.

[0120] In certain embodiments, the methods or compositions described herein are administered in combination with one or more additional therapeutic agents. In certain embodiments, additional therapeutic agents may include anti-inflammatory compounds, anti-angiogenic compounds, anti-fibrotic compounds, or antiproliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNAs, antisense molecules, fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (e.g., methotrexate), or NSAIDs. In certain embodiments, the additional therapeutic agents may include combinations of different classes of therapeutic agents. In certain embodiments, the additional therapeutic agents may be antibody therapies (e.g., belimumab) or corticosteroids.

[0121] This disclosure provides a method for conditioning a subject for immunotherapy (e.g., CAR immunotherapy for the treatment of autoimmune diseases), comprising administering to the subject a preconditioning regimen comprising, or essentially comprising, a dose of cyclophosphamide as described herein. In a relevant aspect, this disclosure provides a method for enhancing the efficacy of immunotherapy (e.g., CAR immunotherapy for the treatment of autoimmune diseases) by conditioning the subject by administering to the subject a preconditioning regimen comprising, or essentially comprising, one or more doses of cyclophosphamide as described herein. While not wishing to be bound by theory, the preconditioning regimens of this disclosure will be understood to enhance the viability, persistence, and efficacy of administered CAR immune cells by, for example, increasing the levels of homeostatic cytokines, e.g., IL-7 and IL-15, and reducing the number of endogenous immune cells competing for these cytokines, thereby creating a more favorable environment for the proliferation of the injected cells. The preconditioning regimens of this disclosure are expected to effectively enable the treatment of autoimmune disorders with cell therapy while minimizing the toxic side effects and risks associated with cyclophosphamide and fludarabine administration. In certain embodiments, the preconditioning regimens of this disclosure, when administered to a subject, are, for example, standard preconditioning regimens (e.g., 1,000 mg / m²). 2 Cyclophosphamide and 25 mg / m² 2 It causes a smaller decrease in white blood cell count compared to fludarabine.

[0122] III. Chimeric Antigen Receptors In addition to the exemplary CARs used in the therapeutics of this disclosure, nucleic acids encoding such CARs, vectors containing such nucleic acids, genetically modified cells that express such CARs, and other related methods and compositions are described in further detail below.

[0123] In certain embodiments, the CAR of the present disclosure comprises (1) an extracellular domain containing an antigen-binding site that provides specificity to a desired antigen (e.g., an immune cell surface antigen), (2) a transmembrane domain, (3) an intracellular signaling domain, and optionally, (4) a costimulatory domain. In certain embodiments, the intracellular signaling domain is or derived from a stimulating molecule, e.g., a T cell activating domain that provides a primary activation signal. Upon specific binding to the targeted antigen, the receptor typically transmits an immunoactivating signal to the cell, e.g., a signal transmitted by ITAM, thereby activating the cell and promoting the targeted immune response. In certain embodiments, the CAR further comprises one or more costimulatory signaling domains, including a functional signaling domain derived from one or more costimulatory molecules. Further examples of CARs are cited in U.S. Patent Nos. 7,446,190 and 9,181,527, U.S. Patent Applications Nos. 2016 / 0340406 and 2017 / 0049819, and International Patent Application Publication No. WO2018 / 140725.

[0124] a. CAR extracellular domain In certain embodiments, the extracellular domain of the CAR includes an antigen-binding site that specifically binds to a target antigen. For example, in certain embodiments, the extracellular antigen-binding domain includes an antigen-binding fragment of an antibody or its derivative. In certain embodiments, the extracellular domain includes a Fab fragment or an scFv. In certain embodiments, the extracellular domain includes an scFv. In certain embodiments, the antigen-binding site is located in the Fab or scFv.

[0125] In certain embodiments, the target antigen specifically bound by the antigen-binding site is a polypeptide. In certain embodiments, the target antigen is selectively expressed or overexpressed on cells of a specific cell type. In certain embodiments, the antigen is expressed by immune cells. In certain embodiments, the targeted antigen is expressed by immune cells, e.g., B cells. In certain embodiments, the targeted antigen is present on the surface of immune cells, e.g., B cells. In certain embodiments, the antigen is a B cell marker. In certain embodiments, the antigen targeted by CAR is CD19, CD20, BCMA, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30. In certain embodiments, the antigen is CD19.

[0126] As described herein, the antigen-binding site of the CAR of this disclosure is (a) CDR H1 -CDR H2 -CDR H3 The immunoglobulin heavy chain variable region (V) containing the structure H ), and (b) CDR L1 -CDR L2 -CDR L3 The immunoglobulin light chain variable region (V) containing the structure L ) may include, where V H and V L These together define a single binding site for binding to the targeted antigen. In a particular embodiment, V H and V L Each includes one or more framework (FR) regions (e.g., one, two, three, or four framework regions). In a particular embodiment, V H FR H1 -CDR H1 -FR H2 -CDR H2 -FR H3 -CDR H3 -FR H4 Includes the structure and / or V L FR L1 -CDR L1 -FR L2-CDR L2 -FR L3 -CDR L -FR L4 It includes the structure.

[0127] Exemplary antigen-binding sites that can bind to CD19 and be used in the CARs of this disclosure are described in more detail below.

[0128] In a particular embodiment, the antigen-binding site that binds to CD19 is V of the antigen-binding sites disclosed in Table 2, 3, or 4. H V containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence H , and V of the same antigen-binding site disclosed in Tables 2, 3, or 4 L V containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence L This includes. In certain embodiments, the antigen-binding site that binds to CD19 may be identified using a CDR determination algorithm known in the art, for example, the algorithm disclosed herein, as shown in SEQ ID NO: 4 V H V of the sequence and sequence number 8 L CDR exists in the array H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 The sequence includes. In a particular embodiment, the antigen-binding site that binds to CD19 can be identified using a CDR determination algorithm known in the art, for example, the algorithm disclosed herein, as shown in SEQ ID NO: 13.H V of sequence and sequence number 17 L CDR exists in the array H 1. CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 The sequence includes. In a particular embodiment, the antigen-binding site that binds to CD19 can be identified using a CDR determination algorithm known in the art, for example, the algorithm disclosed herein, V of SEQ ID NO: 44 H V of sequence and sequence number 48 L CDR exists in the array H 1. CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 The sequence is included. In certain embodiments, the antigen-binding site is a CDR determined based on IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), for example, as shown in Table 2. H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 Includes array, in table, V H and V L The CDR sequence in each sequence is underlined and further indicated individually. In certain embodiments, the antigen-binding site is a CDR determined based on Chothia (see, for example, Chothia C & Lesk AM, (1987), J.Mol.Biol.196:901-917), as shown in Table 3. H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 Includes array, in table, V H and V LThe CDR sequence in each sequence is underlined and further indicated individually. In certain embodiments, the antigen-binding site is a CDR determined based on Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), as shown in Table 4, for example. H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 Includes array, in table, V H and V L The CDR sequence in each sequence is underlined and then shown individually. In certain embodiments, the antigen-binding site is determined based on MacCallum (see MacCallum RM et al., (1996) J.Mol.Biol.262:732-745) or any other CDR determination method known in the art, as of the V of the antibody disclosed in Tables 2, 3, or 4. H and V L CDR of the array H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 This includes sequences. The identification of CDRs and framework sequences is within the normal level of skill in the art, and it will be understood that the boundary between CDRs and framework sequences may depend on the definition or rules used (e.g., IMGT, Kabat, Chothia, etc.).

[0129] In a particular embodiment, the antigen-binding site is the CDR of the antigen-binding site disclosed in Table 2. H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 Includes array, in table, V H and V LThe CDR sequence in each of the sequences is underlined. In a particular embodiment, the antigen-binding site is V of the antigen-binding sites disclosed in Table 2. H and V L Includes arrays.

[0130] [Table 2] TIFF2026514435000004.tif90165

[0131] In a particular embodiment, the antigen-binding site is the CDR of the antigen-binding site disclosed in Table 3. H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 Includes array, in table, V H and V L The CDR sequence in each of the sequences is underlined. In a particular embodiment, the antigen-binding site is V of the antigen-binding site disclosed in Table 3. H and V L Includes arrays.

[0132] [Table 3] TIFF2026514435000006.tif93165

[0133] In a particular embodiment, the antigen-binding site is the CDR of the antigen-binding site disclosed in Table 4. H1 , CDR H2 , CDR H3 , CDR L1 , CDR L2 , and CDR L3 Includes array, in table, V H and V L The CDR sequence in each of the sequences is underlined. In a particular embodiment, the antigen-binding site is V of the antigen-binding site disclosed in Table 4. H and V L Includes arrays.

[0134] [Table 4] TIFF2026514435000008.tif98165

[0135] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in Sequence ID No. 1, 2, and 3, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH and / or CDR sequences described in Sequence IDs 5, 6, and 7, respectively, are positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0136] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs: 10, 11, and 12, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDR described in SEQ ID NOs. 14, 15, and 16, respectively, have sequences positioned between immunoglobulin FR sequences. L1 , CDRL2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0137] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in Sequence ID Nos. 41, 42, and 43, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDR described in SEQ ID NOs. 45, 46, and 47, respectively, whose sequences are positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0138] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs: 30, 31, and 32, respectively.H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDRs described in SEQ ID NOs. 33, 34, and 7, respectively, have sequences positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0139] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs. 36, 37, and 38, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDR described in SEQ ID NOs. 39, 40, and 16, respectively, whose sequences are positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDRH3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0140] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs: 49, 50, and 51, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDR described in SEQ ID NOs. 52, 53, and 47, respectively, whose sequences are positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0141] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs. 54, 55, and 32, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDRs described in SEQ ID NOs. 33, 34, and 7, respectively, have sequences positioned between immunoglobulin FR sequences.L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0142] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs. 56, 57, and 38, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDR described in SEQ ID NOs. 39, 40, and 16, respectively, whose sequences are positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0143] In a particular embodiment, the antigen-binding site that binds to CD19 is the CDR described in SEQ ID NOs. 58, 59, and 51, respectively. H1 , CDR H2 , and CDR H3 V containing an array H And, CDR H1 , CDR H2 and CDR H3 The VH, and / or the CDR described in SEQ ID NOs. 52, 53, and 47, respectively, whose sequences are positioned between immunoglobulin FR sequences. L1 , CDR L2 , and CDR L3 V containing an array L And, CDR L1 , CDR L2 , and CDR L3 The VL includes a sequence positioned between immunoglobulin FR sequences. In certain embodiments, the CDR H1 , CDR H2 , and CDR H3 Array, and / or CDR L1 , CDR L2 , and CDR L3 The sequences are positioned between human or humanized immunoglobulin FR sequences.

[0144] In a particular embodiment, the antigen-binding site that binds to CD19 is V, which includes the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13, or SEQ ID NO: 44. H Includes. In certain embodiments, the antigen-binding site that binds to CD19 includes the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 17, or SEQ ID NO: 48. L Includes.

[0145] In a particular embodiment, the antigen-binding site that binds to CD19 is V, which contains the amino acid sequence of SEQ ID NO: 4. H , and / or V containing the amino acid sequence of SEQ ID NO: 8 L Includes.

[0146] In a particular embodiment, the antigen-binding site that binds to CD19 is V containing the amino acid sequence of SEQ ID NO: 13. H, and / or V containing the amino acid sequence of SEQ ID NO: 17 L Includes.

[0147] In a particular embodiment, the antigen-binding site that binds to CD19 is V, which contains the amino acid sequence of SEQ ID NO: 44. H , and / or V containing the amino acid sequence of SEQ ID NO: 48 L Includes.

[0148] In a particular embodiment, the antigen-binding site that binds to CD19 contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 44. H This includes, or in addition, the antigen-binding site that binds to CD19 contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 17, and SEQ ID NO: 48. L Includes.

[0149] In a particular embodiment, the antigen-binding site that binds to CD19 contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: V H V contains and / or contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of Sequence ID No. 8. L Includes.

[0150] In a particular embodiment, the antigen-binding site that binds to CD19 contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected from SEQ ID NO: 13. HV contains and / or contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of Sequence ID No. 17. L Includes.

[0151] In a particular embodiment, the antigen-binding site that binds to CD19 contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence selected from SEQ ID NO: 44 H V contains and / or contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of Sequence ID No. 48. L Includes.

[0152] In each of the embodiments described above, V is coupled to CD19. H Array and / or V LEach array is independent of V H and / or V L It is intended herein that the framework region may contain amino acid mutations (e.g., substitutions, deletions, or additions of at least 1, 2, 3, 4, 5, or 10 amino acids). In certain embodiments, V which binds together with CD19 H Array and / or V L Each array is independent of V H and / or V L The framework region may contain mutations (e.g., substitutions, deletions, or additions) of 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 amino acids. In a particular embodiment, V H The framework domain is the humanization or human framework domain. In a particular embodiment, V L The framework domain is humanization or the human framework domain.

[0153] In certain embodiments, the antigen-binding site that binds to CD19 is located in the scFv. In certain embodiments, the scFv contains the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the scFv contains the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the scFv contains the amino acid sequence of SEQ ID NO: 60.

[0154] In a particular embodiment, the antigen-binding site for CD19 is located in the scFv, where the scFv contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 9.

[0155] In a particular embodiment, the antigen-binding site for CD19 is located in the scFv, where the scFv contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from SEQ ID NO: 18.

[0156] In a particular embodiment, the antigen-binding site for CD19 is located in the scFv, where the scFv contains an amino acid sequence that is at least 60% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an amino acid sequence selected from SEQ ID NO: 60.

[0157] Further examples of antigen-binding sites that bind to CD19 include U.S. Patent Nos. 7,446,179, 9,765,156, 10,125,193, 10,221,245, 10,287,350, 10,301,388, 10,457,730, 10,493,139, 10,533,055, 10,662,248, 10,780,118, and 10, Patent Nos. 844,120, 10,874,693, 11,001,639, 11,034,750, 11,034,763, 11,077,144, and 11,141,436; U.S. Patent Applications Nos. 2020 / 0038443, 2020 / 0062843, 2020 / 0123254, 2020 / 0289563, 2020 / 0376033, and Issues 2020 / 0384023, 2020 / 0384026, 2021 / 0002366, 2021 / 0061907, 2021 / 0069244, 2021 / 0101978, 2021 / 0196756, 2021 / 0238253, 2021 / 0332133, 2021 / 0395362, and 2021 / 0395364; and International This is published in Patent Application Publications No. 2018 / 201794, 2019 / 137518, 2019 / 154313, 2019 / 214332, 2020 / 233589, 2021 / 170146, 2021 / 217130, 2021 / 223719, 2021 / 223720, 2021 / 225532, and 2022 / 012683.

[0158] In certain embodiments, the antigen-binding site is measured using a standard binding assay, such as surface plasmon resonance or biolayer interferometry, with K levels of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM, so as to be measured using a standard binding assay, such as surface plasmon resonance or biolayer interferometry. D, or bind to CD19 with a stronger KD. In a particular embodiment, the antigen-binding site is measured using a standard binding assay, such as surface plasmon resonance or biolayer interferometry, in the following ranges: approximately 20 nM to approximately 0.05 nM, approximately 20 nM to approximately 0.075 nM, approximately 20 nM to approximately 0.1 nM, approximately 20 nM to approximately 0.5 nM, approximately 20 nM to approximately 1 nM, approximately 10 nM to approximately 0.05 nM, approximately 10 nM to approximately 0.075 nM, approximately 10 nM to approximately 0.1 nM, approximately 10 nM to approximately 0.5 nM, approximately 10 nM to approximately 1 nM, approximately 5 nM to approximately 0.05 nM, approximately 5 nM to approximately 0.075 nM, approximately 5 nM to approximately 0.1 nM, approximately 5 nM to approximately 0.5 nM, approximately 5 nM to approximately 1 nM, approximately 3 nM to approximately 0.05 nM, approximately 3 nM to approximately 0.05 nM. Approximately 0.075nM, approximately 3nM to approximately 0.1nM, approximately 3nM to approximately 0.5nM, approximately 3nM to approximately 1nM, approximately 3nM to approximately 2nM, approximately 2nM to approximately 0.05nM, approximately 2nM ~about 0.075nM, about 2nM to about 0.1nM, about 2nM to about 0.5nM, about 2nM to about 1nM, about 1nM to about 0.05nM, about 1nM to about 0.075nM, K in the ranges of approximately 1 nM to 0.1 nM, 1 nM to 0.5 nM, 0.5 nM to 0.05 nM, 0.5 nM to 0.075 nM, 0.5 nM to 0.1 nM, 0.1 nM to 0.05 nM, 0.1 nM to 0.075 nM, or 0.075 nM to 0.05 nM, or 0.05 nM to 0.035 nM D Then combine it with CD19.

[0159] In certain embodiments, the antigen-binding site that binds to CD19 cross-competes with the antigen-binding sites disclosed in Tables 2, 3, or 4. Competition assays for determining whether an antigen-binding site binds to the same epitope as the disclosed antibody, or whether it competes with the disclosed antibody for binding, are known in the Art. Exemplary competition assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry.

[0160] The antigen-binding sites disclosed herein may be further optimized (e.g., affinity maturation) to improve biochemical properties, including affinity and / or specificity; to improve biophysical properties, including aggregation, stability, precipitation, and / or nonspecific interactions; and / or to reduce immunogenicity. Affinity maturation methods are within the scope of common art in the art. For example, diversity may be introduced into immunoglobulin heavy chains and / or immunoglobulin light chains by DNA shuffling, strand shuffling, CDR shuffling, random mutagenesis, and / or site-directed mutagenesis.

[0161] Generally, an optimized antigen-binding site has at least the same or substantially the same affinity for the antigen as the unoptimized (or parent) antigen-binding site from which it originated. Preferably, the optimized antibody has a higher affinity for the antigen compared to the parent antibody.

[0162] The functional ability of a CAR to specifically bind to its target antigen (e.g., CD19) can be evaluated in the Jurkat reporter cell line, where CAR activation depends on binding to the target protein bound to the plate or to the target protein bound to the cell (in response, activated cells emit green fluorescence due to the NFAT-GFP reporter construct they contain). This method is a useful and reliable qualitative measurement of functional binding ability.

[0163] In certain embodiments, the extracellular binding domain of the CAR includes means for binding to CD19. In certain embodiments, the means is an antigen-binding site, for example, an antigen-binding site that binds to CD19 as described herein.

[0164] b. CAR transmembrane domain The extracellular antigen-binding domain can be fused to the transmembrane domain of the CAR. In certain embodiments, the transmembrane domain of the CAR is derived from a naturally occurring transmembrane protein. In certain embodiments, the transmembrane domain can signal to the intracellular domain(s) whenever the CAR binds to a target. In some embodiments, the transmembrane domain(s) may be TCRα, TCRβ, TCRζ, CD28, CD3ε, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, EGFR, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1(CD11a, CD18), ICOS(CD2 78), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, C D160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, C D11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD22 9) The transmembrane domain comprises one or more transmembrane regions of proteins selected from the group consisting of CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D, and NKG2C. In certain embodiments, the transmembrane domain comprises one or more transmembrane regions of proteins selected from the group consisting of CD8α, CD28, CD3ζ, and CD4. In certain embodiments, the transmembrane domain comprises one or more transmembrane regions of proteins selected from the group consisting of CD8α and CD28.In certain embodiments, the transmembrane domain includes the CD8α transmembrane domain. In certain embodiments, the transmembrane domain is one that naturally associates with one of the domains of the CAR (e.g., the primary signaling domain or the co-stimulatory signaling domain). In certain embodiments, the transmembrane domain may be selected or modified by amino acid substitution to avoid multimerization with the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex (e.g., the CAR complex). In other embodiments, the transmembrane domain is capable of homodimerization with another CAR on the surface of an immune cell (e.g., a T cell). In certain embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present on the same immune cell.

[0165] In certain embodiments, the transmembrane domain includes a CD8α transmembrane domain. In certain embodiments, the transmembrane domain includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain includes the amino acid sequence of SEQ ID NO: 19.

[0166] In certain embodiments, the transmembrane domain includes conserved substitutions compared to the transmembrane domains disclosed herein, for example, compared to the transmembrane domain containing the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain includes fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved substitutions compared to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conserved substitutions compared to the amino acid sequence of SEQ ID NO: 19.

[0167] In certain embodiments, the transmembrane domain includes the CD28 transmembrane domain. In certain embodiments, the transmembrane domain includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the transmembrane domain includes the amino acid sequence of SEQ ID NO: 61.

[0168] In certain embodiments, the transmembrane domain includes conserved substitutions compared to the transmembrane domains disclosed herein, for example, compared to the transmembrane domain containing the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the transmembrane domain includes fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved substitutions compared to the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the transmembrane domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conserved substitutions compared to the amino acid sequence of SEQ ID NO: 61.

[0169] In certain embodiments, the CAR comprises a transmembrane domain and / or a cytoplasmic (intracellular) domain derived from the killer immunoglobulin-like receptor (KIR) family of proteins. The KIR gene family comprises at least 15 loci (KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3) and two pseudogenes (KIR2DP1 and KIR3DP1) encoded within a 100-200Kb region of the leukocyte receptor complex (LRC) located on chromosome 19 (19q13.4). The LRC constitutes a large, 1Mb high-density cluster of rapidly evolving immunogenes, which includes genes encoding other cell surface molecules having characteristic Ig-like extracellular domains. In addition, the extended LRC contains genes encoding the transmembrane adapter molecules DAP10 and DAP12. Thus, in certain embodiments, a cell containing the CAR of this disclosure, which includes a KIR transmembrane domain and / or cytoplasmic domain, may also contain a polynucleotide encoding DAP10 or DAP12. In certain embodiments, the KIR is KIRS2 or KIR2DS2.

[0170] c.CAR intracellular domain The intracellular domain of a CAR includes an intracellular signaling domain (i.e., a functional signaling domain derived from a stimulating molecule) and optionally one or more co-stimulatory signaling domains (i.e., a functional signaling domain derived from at least one co-stimulatory molecule). These intracellular and co-stimulatory domains are at least partially responsible for immune cell responses, including, but are not limited to, the proliferation, differentiation, and activation of specific functions (e.g., T cell cytotoxicity or cytokine secretion) of immune cells on which the CAR is expressed. Therefore, the intracellular signaling domain is intended to encompass any cleaved portion of the intracellular signaling domain sufficient to transmit effector functional signals.

[0171] The stimulative intracellular signaling domain may contain signaling motifs known as immune receptor-activated tyrosine motifs (ITAMs). Examples of cytoplasmic signaling sequences containing ITAMs particularly useful in the CARs of this application include those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In certain embodiments, the intracellular signaling domain of the CAR described herein includes a functional cytoplasmic signaling domain derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD28, CD79a, CD79b, CD66d, 4-1BB, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), DAP10, and / or DAP12. In certain embodiments, the intracellular signaling domain includes a CD3ζ signaling domain or an FcRγ signaling domain.

[0172] In certain embodiments, the CAR of this disclosure comprises a CD3ζ signaling domain, either alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the CAR may comprise a CD3ζ chain portion (i.e., a CD3ζ intracellular signaling domain) and an intracellular domain of a co-stimulatory molecule, e.g., a 4-1BB intracellular domain. In certain embodiments, the CD3ζ intracellular signaling domain is the intracellular domain of human T cell surface glycoprotein CD3ζ chain isoform 3 (human CD247). The human CD3ζ intracellular domain, without HLA restriction, evokes stimulating intracellular signaling upon binding of an extracellular antigen to its ligand.

[0173] In certain embodiments, the CAR of the Disclosure comprises a CD3ζ signaling domain. In certain embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 21.

[0174] In certain embodiments, the intracellular signaling domain includes conserved substitutions compared to the intracellular signaling domains disclosed herein, for example, compared to the intracellular signaling domain including the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain includes fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved substitutions compared to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conserved substitutions compared to the amino acid sequence of SEQ ID NO: 21.

[0175] In certain embodiments, the CAR of the Disclosure includes a co-stimulatory domain. The co-stimulatory domain includes a functional signaling domain derived from a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an efficient response of lymphocytes to an antigen. Examples of co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, Lymphocyte function-associated antigen 1 (LFA-1, CD11a / CD18), CD2, CD7, CD258 (LIGHT), NKG2C, B7-H3, CD83 ligand, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, and CD1 Examples include 03, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, and PAG / Cbp. In certain embodiments, the co-stimulatory domain of CAR includes a functional signaling domain of a co-stimulatory molecule described herein, such as OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, CD83 ligand, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), or any combination thereof.In certain embodiments, the co-stimulatory domain of CAR includes a functional signaling domain of a co-stimulatory molecule selected from 4-1BB(CD137), CD28, ICOS, CD27, CD40, and OX40. In certain embodiments, CAR includes a co-stimulatory domain comprising a 4-1BB intracellular domain and / or a CD28 intracellular domain.

[0176] In certain embodiments, the CAR of the Disclosure comprises a co-stimulatory domain including a 4-1BB intracellular domain. In certain embodiments, the co-stimulatory domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 20.

[0177] In certain embodiments, the co-stimulatory domain includes conserved substitutions compared to the co-stimulatory domains disclosed herein, for example, compared to the co-stimulatory domain containing the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the co-stimulatory domain includes fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions compared to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions compared to the amino acid sequence of SEQ ID NO: 20.

[0178] In certain embodiments, the CAR of this disclosure includes a co-stimulatory domain containing a CD28 intracellular domain. In certain embodiments, the co-stimulatory domain includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 62.

[0179] In certain embodiments, the co-stimulatory domain includes conserved substitutions compared to the co-stimulatory domains disclosed herein, for example, compared to the co-stimulatory domain containing the amino acid sequence of SEQ ID NO: 62. In certain embodiments, the co-stimulatory domain includes fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions compared to the amino acid sequence of SEQ ID NO: 62. In certain embodiments, the intracellular signaling domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conservative substitutions compared to the amino acid sequence of SEQ ID NO: 62.

[0180] The intracellular signaling and co-stimulatory domains within the cytoplasmic portion of the CAR can be linked to each other in a random or specific order. In certain embodiments, the co-stimulatory signaling domain is located at the N-terminus of the primary signaling domain. Optionally, the intracellular domains are linked by short oligopeptide or polypeptide linkers, for example, 2–10 amino acids in length. In certain embodiments, the intracellular domains are linked by, for example, a GS doublet linker or (G4S) nThey are linked by a linker. In certain embodiments, the intracellular signaling domain (e.g., the CD3ζ signaling domain) is located in the C-terminal domain of the CAR.

[0181] d. Other CAR domains The extracellular antigen-binding domain of a CAR may be linked to the transmembrane domain by a hinge domain or linker. In certain embodiments, the hinge domain or linker is positioned between the antigen-binding site and the transmembrane domain. A variety of hinges or linkers may be used, but are not limited to, human Ig (immunoglobulin) hinges (e.g., IgG4 hinge, IgD hinge), Gly-Ser linker, (G4S)4 linker, KIR2DS2 hinge, and CD8α hinge.

[0182] In certain embodiments, the hinge domain is the CD8α hinge. In certain embodiments, the hinge domain contains an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the hinge domain contains the amino acid sequence of SEQ ID NO: 22.

[0183] In certain embodiments, the hinge domain includes conserved substitutions compared to hinge domains disclosed herein, for example, compared to a hinge domain containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the hinge domain includes fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved substitutions compared to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the hinge domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more conserved substitutions compared to the amino acid sequence of SEQ ID NO: 22.

[0184] In certain embodiments, the CAR includes a CD8α transmembrane domain and a CD8α hinge.

[0185] In certain embodiments, CAR comprises a signal peptide. In certain embodiments, the nucleic acid encoding CAR comprises a nucleic acid sequence encoding a signal peptide. In certain embodiments, the signal peptide is derived from a native polypeptide. In other embodiments, the signal peptide comprises a heterologous or non-native signal peptide. In certain embodiments, the signal peptide is a CD8α signal peptide or an IgG signal peptide.

[0186] In certain embodiments, CAR comprises a CD8α signal peptide. In certain embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 25. In certain embodiments, CAR comprises an IgG signal peptide. In certain embodiments, the signal peptide contains an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 26.

[0187] In certain embodiments, the signal peptide includes conserved substitutions compared to the signal peptide disclosed herein, for example, a signal peptide containing the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments, the signal peptide includes fewer than two, three, four, five, six, seven, eight, nine, or ten conserved substitutions compared to the signal peptide of SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments, the signal peptide includes one, two, three, four, five, six, seven, eight, nine, or ten or more conserved substitutions compared to the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26.

[0188] e. Exemplary CAR structures In certain embodiments, the CAR of the Disclosure comprises, in the N-terminal to C-terminal direction: (1) an extracellular domain comprising an antigen-binding site, e.g., an scFv, e.g., an anti-CD19 scFv, e.g., an scFv comprising the amino acid sequence of SEQ ID NO: 9; (2) an optional hinge domain or linker, e.g., a CD8α hinge, e.g., a hinge comprising the amino acid sequence of SEQ ID NO: 22; (3) a transmembrane domain, e.g., a CD8α transmembrane domain, e.g., a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 19; (4) an optional co-stimulatory domain, e.g., an intracellular domain of 4-1BB, e.g., a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 20; and (e) an intracellular signaling domain, e.g., a CD3ζ signaling domain, e.g., a signaling domain comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the CAR further comprises an N-terminal signal peptide, e.g., a CD8 signal peptide, e.g., a signal peptide comprising the amino acid sequence of SEQ ID NO: 25. A particular exemplary anti-CD19 CAR construct of the Disclosure is shown in Figure 1.

[0189] In certain embodiments, the CAR of the Disclosure includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the CAR of the Disclosure includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the CAR of the Disclosure includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 63.In certain embodiments, the CAR of the Disclosure includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 64.

[0190] In certain embodiments, the CARs of this disclosure include conserved substitutions compared to the CARs disclosed herein, for example, a CAR containing the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 63, or SEQ ID NO: 64. In certain embodiments, the CARs include fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 conserved substitutions compared to the CARs of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 63, or SEQ ID NO: 64. In certain embodiments, the CAR includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, or thirty or more conservative substitutions compared to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 63, or SEQ ID NO: 64.

[0191] In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0192] A vector containing a polynucleotide encoding f.CAR In certain embodiments, the CAR used in the methods of the present disclosure is encoded by a polynucleotide, which may optionally be present in a vector. In certain embodiments, the polynucleotide encodes a CAR comprising an extracellular domain including an antigen-binding site, a transmembrane domain, an intracellular signaling domain, and optionally a costimulatory domain. In certain embodiments, the polynucleotide encodes a CAR comprising an anti-CD19 scFv, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0193] In certain embodiments, the vectors include plasmid vectors, viral vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, AAVs, retrotransposons (e.g., piggyback, Sleeping Beauty), site-directed insertion vectors (e.g., CRISPR, zinc finger nucleases, TALENs), or suicide expression vectors, or other vectors known in the art.

[0194] In certain embodiments, the vector is a viral vector, for example, a lentiviral vector, for example, a third-generation lentiviral vector, for example, a third-generation self-inactivating lentiviral vector.

[0195] In certain embodiments, the vector is an RNA vector, such as an mRNA vector. This may achieve the same therapeutic effect as in cells transduced with a virus, but it will not be permanent because the mRNA is diluted with cell division. Depending on the case, the RNA vector may be introduced into the host cell by electroporation, or by lipid nanoparticles (LNPs), such as targeted LNPs.

[0196] CAR expression can be confirmed by sequencing. Full-length CAR protein expression can be confirmed using immunoblotting, immunohistochemistry, flow cytometry, or other techniques known and available in the art.

[0197] This disclosure also provides vectors into which DNA or RNA encoding the CAR of this disclosure has been inserted. Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of the transgene and its transmission in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as mouse leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of resulting in low immunogenicity in the target into which they are introduced.

[0198] In short, the expression of native or synthetic polynucleotides encoding CARs is typically achieved by operably ligating a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector is generally replicable in mammalian cells and / or can be incorporated into the mammalian cell genome. Typical vectors contain transcriptional and translational terminators, start sequences, and promoters useful for regulating the expression of desired nucleic acid sequences.

[0199] Nucleic acids can be cloned into any number of different types of vectors. For example, nucleic acids can be cloned into vectors that include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0200] Expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, as well as in other manuals on virology and molecular biology. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a replication origin, promoter sequence, convenient restriction endonuclease site, and one or more selection markers that function in at least one organism (e.g., WO2001 / 96584, WO2001 / 29058, and U.S. Patent No. 6,326,193).

[0201] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, but in recent years, many promoters have been shown to also contain functional elements downstream of the initiation site. Often, there is flexibility in the spacing between promoter elements, and as a result, promoter function is maintained even if the elements are inverted or moved relative to each other. In some promoters, individual elements appear to be able to activate transcription cooperatively or independently.

[0202] An example of a promoter that can be used is the cytomegalovirus (CMV) initial promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can activate high levels of expression of any functionally linked polynucleotide sequence. Other constitutive promoter sequences may also be used as needed, but are not limited to, the Simian virus 40 (SV40) initial promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus initial promoter, Roussarcoma virus promoter, elongation factor 1α promoter (EF-1α), and human gene promoters, including, but not limited to, actin promoters, myosin promoters, or hemoglobin promoters.

[0203] Furthermore, this disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also envisioned to be used in the methods and compositions of this disclosure. The use of an inducible promoter provides a molecular switch that can turn on the expression of a functionally linked polynucleotide sequence when such expression is desired, or turn off when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In some embodiments, the inducible promoter is activated in response to an extracellular ligand. For example, in some embodiments, the inducible promoter is activated by the binding of an extracellular ligand to a synthetic receptor (and the expression of the CAR is regulated). For example, in some embodiments, a synthetic receptor, such as a synthetic Notch receptor (i.e., "synNotch"), may be used as a binding-induced transcription switch that activates a promoter to which a nucleic acid sequence encoding a CAR is operably linked when it binds to its ligand. Therefore, as a non-limiting example, such a system may require the presence of a ligand (e.g., to which synNotch binds) for immune cells to respond to a BCR (e.g., to which CAR binds) or autoantibodies. The requirement of a specific combination to produce a particular signaling output in a molecular circuit is linked to a logic gate. See, for example, Roybal et al., 2016 Cell 164(4):770-9.

[0204] Examples of other systems for expressing or regulating the expression of chimeric receptors include Wu et al. (2015) Science 350:aab4077, Fedorov et al. (2014) Cancer Journal 20:160-165, Kloss et al. (2013) Nature Biotechnology 31:71-75, Sakemura et al. (2016) Cancer Immunol.Res.4:658-668, Hill et al. (2018) Nat.Chem.Biol.14:112-117, Di Stasi et al. (2011) N.Engl.J.Med.365:1673-1683, Budde et al. (2013) PLoS One 8:e82742, Wei et al. (2012) Nature 488:384-388, Ma et Examples include those described in al. (2016) Proc.Natl.Acad.Sci. USA 113:E450-458, Rodgers et al. (2016) Proc.Natl.Acad.Sci.USA 113:E459-468, Kudo et al. (2014) Cancer Res.74:93-103, and Chen et al. (2010) Proc.Natl.Acad.Sci.USA 107:8531-8536.

[0205] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population intended to be transfected or infected with the viral vector. The selection marker may be contained in a separate DNA or RNA fragment and may be used in a co-transfection procedure. Both the selection marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0206] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is absent from or not expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is revealed by several easily detectable characteristics, such as enzymatic activity. Reporter gene expression is evaluated at an appropriate time after the DNA or RNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with a minimal 5' facile region exhibiting the highest expression level of the reporter gene is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used to evaluate the action of the agent for its ability to regulate promoter-driven transcription.

[0207] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0208] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particulate guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY.

[0209] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. RNA vectors include those having an RNA promoter and other relevant domains for the production of RNA transcripts. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0210] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, which include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery media in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). Other methods of targeted delivery of nucleic acids are known, such as delivery of polynucleotides by targeted nanoparticles or other suitable submicron-sized delivery systems.

[0211] When nonviral delivery systems are used, exemplary delivery media are liposomes or lipid nanoparticles (LNPs). Lipid formulations are intended for the (in vitro, ex vivo, or in vivo) delivery of nucleic acids to host cells. In another embodiment, nucleic acids may be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both liposomes and oligonucleotides, contained within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained in lipids as suspensions, contained in micelles, complexed with them, or otherwise associated with lipids. Compositions of associated lipids, lipid / DNA, lipid / RNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles, or as "disintegrated" structures. They may also simply be scattered in solution, perhaps forming aggregates of non-uniform size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. Examples of lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds including long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Examples of lipids, liposomes, lipid nanoparticles, and related formulations are, for example, U.S. Patents 8,058,069, 8,492,359, 8,822,668, 9,006,417, 9,139,554, 9,364,435, 9,404,127, 9,415,109, 9,504,651, 9,518,272, and 9,5 No. 67,296, No. 9,580,711, No. 9,636,414, No. 9,694,077, No. 9,758,795, No. 9,814,777, No. 9,868,692 , No. 9,878,042, No. 9,943,846, No. 9,950,065, No. 10,041,091, No. 10,106,490, No. 10,166,298, 10,221,127, 10,227,302, 10,233,148, 10,266,485, 10,442,756, 10,485,884, 10,561,732, 10, No. 576,146, No. 10,577,403, No. 10,653,780, No. 10,703,789, No. 10,980,895, No. 11,045,418, No. 11,141,378, No. 11,1 Nos. 73,120, 11,191,849, 11,285,222, 11,357,856, 11,446,383, 11,453,639, 11,478,552, 11,559,587, U.S. Patent Application Nos. 2011 / 0117125, 2012 / 0264810, 2018 / 0000953, 2018 / 0085474, 2018 / 0185516, 2019 / No. 0022247, No. 2019 / 0032087, No. 2019 / 0274968, No. 2019 / 0336608, No. 2020 / 0046830, No. 2020 / 0109113, No. 2020 / 01 No. 55671, No. 2020 / 0163878, No. 2020 / 0164038, No. 2020 / 0172472, No. 2020 / 0297634, No. 2020 / 0297870, No. 2020 / 03061 This is described in Patent Nos. 91, 2021 / 0145982, 2021 / 0207140, 2021 / 0220274, 2021 / 0346306, 2022 / 0000778, 2022 / 0001029, 2022 / 0118112, and 2022 / 0160899; and in International Patent Application Publications 2008 / 042973, 2021 / 231929, and 2021 / 237084.

[0212] Suitable lipids for use can be obtained from private suppliers. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo., diacetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY), cholesterol ("Chol") can be obtained from Calbiochem-Behring, and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Storage solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid media formed by the formation of closed lipid bilayers or aggregates. Liposomes may be characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement, followed by the formation of a closed structure that encapsulates water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from the usual vesicular structure in solution are also included. For example, lipids may take on a micelle structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also intended.

[0213] In certain embodiments where nonviral delivery methods are utilized, exemplary delivery media are bioabsorbable silicon nanoparticles. Silicon nanoparticles may be prepared from either pure silicon or hydrolyzable silicon-containing materials and may be made porous by standard techniques, e.g., by contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and passing an electric current through them. Silicon nanoparticles may be loaded with polynucleotides, such as RNA, to be delivered to host cells (e.g., in vitro, ex vivo, or in vivo). Silicon nanoparticles may be surface-treated with lipids (e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), stearylamine (SA), and / or lecithin) that may help control the release rate of the payload polynucleotide. Lipid-treated silicon nanoparticles may be further treated with amino acids (e.g., arginine, histidine, and / or glycine) that may promote the stability of the payload nucleic acid, such as RNA. Examples of silicon nanoparticles are described, for example, in U.S. Patent No. 9,132,083, U.S. Patent Applications No. 2022 / 0183989 and 2022 / 0184038, and International Patent Application Publication No. 2020 / 193999.

[0214] Any domain and / or fragment of the CAR, vector, and promoter may be synthetic gene fragments amplified by PCR or any other means known in the art.

[0215] IV. Pharmaceutical Compositions For therapeutic use, the agents (e.g., cyclophosphamide) or cells disclosed herein are preferably present in a pharmaceutical composition, which optionally includes a pharmaceutically acceptable carrier.

[0216] a. Compositions containing cyclophosphamide In certain embodiments, cyclophosphamide is preferably present in the pharmaceutical composition. In certain embodiments, the pharmaceutical composition may contain, for example, formulation materials for modifying, maintaining, or preserving the pH, molar osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, absorption, or penetration of the composition.In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); and low molecular weight polymers. Lipeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic acid, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyroxapole); stability enhancers (e.g., sucrose or sorbitol); isotonic enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery medium; diluents; excipients and / or pharmaceutical adjuvants (e.g., Adeboye (See Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020)).

[0217] In certain embodiments, the pharmaceutical composition may contain nanoparticles, such as polymer nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) Bioeng.Transl.Med.1:10-29).

[0218] In certain embodiments, the pharmaceutical composition may contain sustained-release or controlled-release formulations. Techniques for constructing sustained-release or controlled-release means, such as liposome carriers, biodegradable microparticles or porous beads, and accumulative injections, are also known to those skilled in the art. Examples of sustained-release formulations include molded articles, such as films or microcapsules containing porous polymer microparticles or semipermeable polymer matrices. Examples of sustained-release matrices include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art.

[0219] The pharmaceutical compositions disclosed herein may be provided in unit dosage forms and may be prepared by any preferred method. The pharmaceutical compositions must be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV) administration, endothelial administration, inhalation administration, transdermal administration, topical administration, transmucosal administration, subarachnoid administration, and rectal administration. In certain embodiments, the agents disclosed herein (e.g., cyclophosphamide) are administered by IV infusion. Useful formulations may be prepared by methods known in the pharmaceutical field. See, for example, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020). Suitable formulation components for parenteral administration include sterile diluents, such as sterile water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffering agents, such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0220] Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under manufacturing and storage conditions and protected from microorganisms. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof.

[0221] Intravenous drug delivery formulations may be contained in syringes, pens, or bags. In certain embodiments, the bag may be connected to a channel including a tube and / or needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried and contained in one or more vials. In certain embodiments, freeze-dried formulations in one or more vials may be combined to obtain a therapeutic dose of the drug.

[0222] In certain embodiments, the pharmaceutical composition may contain a stabilizer. In certain embodiments, the stabilizer is a cation, for example, a divalent cation. In certain embodiments, the cation is calcium or magnesium. The cation may be in the form of a salt, for example, calcium chloride (CaCl2) or magnesium chloride (MgCl2).

[0223] In certain embodiments, the pharmaceutical formulation is sterile. Sterilization can be achieved by any preferred method, for example, filtration through a sterile filtration membrane. If the composition is lyophilized, filtration sterilization can be performed before or after lyophilization and reconstitution.

[0224] The compositions described herein may be administered topically or systemically. In preferred embodiments, the pharmaceutical compositions are administered subcutaneously, and in more preferred embodiments, intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0225] The therapeutically effective dose of the drug administered depends on variables such as the type and severity of the disease or indication being treated, the patient's overall health status, the drug's in vivo potency, the pharmaceutical formulation, and the route of administration. The initial dose may be increased beyond the upper limit to rapidly achieve the desired blood or tissue level. Alternatively, the initial dose may be lower than the optimal dose, and the daily dose may be gradually increased throughout the course of treatment. The frequency of administration may vary depending on factors such as the route of administration, the dose, and the disease being treated. The preferred route of administration is intravenous infusion.

[0226] b. Immune cell therapy composition As described above, the genetically modified immune cells of this disclosure are useful in immunotherapy (e.g., adoptive immunotherapy for the treatment of autoimmune diseases). Accordingly, this disclosure provides compositions comprising one or more genetically modified immune cells as described herein. In certain embodiments, one or more genetically modified immune cells are present in a pharmaceutical composition, where, for example, the composition comprises a pharmaceutically acceptable carrier.

[0227] In certain embodiments, the immunotherapy is autologous, meaning that immune cells collected from a patient are cultured in vitro and then administered to the same patient. In certain embodiments, the immunotherapy is allogeneic, meaning that immune cells are collected from a healthy donor, where optionally, the immune cells (e.g., T cells) are genetically engineered to inactivate or lack expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA) molecule, such as an HLA class I component or an HLA class II component. For example, in certain embodiments, allogeneic T cells may be genetically engineered to have reduced or no expression of the functional TCR on their surface, or to have reduced or no expression of one or more subunits constituting the functional TCR (e.g., TCRα chain or TCRβ chain). Alternatively, T cells may express a functionally impaired TCR, for example, by the expression of one or more mutant or cleaved subunits of the TCR. In certain embodiments, allogeneic T cells can be genetically engineered to reduce or eliminate the expression of functional HLA molecules on their surface, for example, by reducing or eliminating the expression of HLA class I molecules or HLA class II molecules. In certain embodiments, surface expression of HLA class I molecules in allogeneic T cells is reduced by targeting or knocking out a sequence encoding β2 microglobulin (β2M). In certain embodiments, surface expression of HLA class II molecules in allogeneic T cells is reduced by targeting or knocking out a sequence encoding CIITA. Such cells can be produced using a gene editing system as described herein. In embodiments, a gene editing system targeting sequences encoding TCRα, TCRβ, β2M, and / or CIITA is introduced into cells so as to downregulate the surface expression of functional TCRs, HLA class I molecules, and / or HLA class II molecules. In some embodiments, allogeneic T cells may lack a functional TCR and functional HLA molecules, such as HLA class I molecules and / or HLA class II molecules.

[0228] Immunotherapy can be provided as a cell composition. In certain embodiments, the genetically engineered immune cells in the composition are NK cells, e.g., CAR NK cells. In certain embodiments, the genetically engineered immune cells in the composition are T cells, e.g., CAR T cells. It will be understood that other types of cells, such as APCs, may be used to proliferate T cells ex vivo. Thus, the cell composition may contain other cell types in addition to T cells. In certain embodiments, the cell composition is enriched with T cells. If T cells are prepared by stimulation with APCs in ex vivo cell culture, the T cells may be enriched by methods known in the art. For example, in certain embodiments, APCs are removed from the cell culture by surface marker-based magnetic bead selection or cell sorting. In certain embodiments, APCs are outgrown by T cells under conditions that favor T cell proliferation (e.g., cytokines). In certain embodiments, APCs are removed by stronger adhesion of APCs to tissue culture plates than T cells. Concentration can produce compositions in which at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the composition are T cells. In certain embodiments, the composition is substantially free of myeloid cells. For example, in certain embodiments, the percentage of myeloid cells relative to all cells in the composition is 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0229] In certain embodiments, the immune cell composition may comprise one or more immunogenicity-enhancing adjuvants (also referred to herein as “adjuvants”). Such adjuvants are substances that enhance or strengthen an immune response (e.g., an immune response to an antigen mediated by CD8-positive T cells and helper T(TH) cells) in a non-antigen-specific manner and are therefore considered useful in the pharmaceutical compositions disclosed herein. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligands, FLT3 ligands, GM-CSF, IC30, IC31, imiquimod (ALDARA®), regiquimod, IMUFACT®, IMP321, interleukins such as IL-2, IL-13, IL-21, interferon-α or -β, or their pegylated derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, JUVIMMUNE®, LIPOVAC®, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide Examples include ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK®, OspA, poly(lactidocoglycolide)[PLG]-based microparticles and dextran microparticles, talactoferrin, SRL 172, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon (derived from saponins, mycobacterial extracts, and synthetic bacterial cell wall mimics), and other registered trademark adjuvants, such as Ribi's Detox, QUIL®, or Superfos. Depending on the circumstances, adjuvants such as Freund's adjuvant or GM-CSF may also be preferred. Several dendritic cell-specific immunoadjuvants (e.g., MF59) and their preparation methods have been previously described (Ott et al. (1995) Pharm Biotechnol. 6:277-96).In certain embodiments, the adjuvant is a naturally occurring adjuvant. In certain embodiments, the adjuvant is a non-natural adjuvant.

[0230] The immunotherapy composition may further comprise one or more carriers and / or excipients. Exemplary carriers and excipients are described herein (see the subsection “Compositions Containing Cyclophosphamide and / or Fludarabine” above).

[0231] The therapeutically effective dose of genetically modified immune cells administered may depend on variables such as the type and severity of the disease or indication being treated, the patient's overall health status, the in vivo potency of the drug, the pharmaceutical formulation, and the route of administration. The preferred route of administration is intravenous infusion.

[0232] Throughout this description, where a composition is described as having, encompassing, or containing a particular component, or where a process and method is described as having, encompassing, or containing a particular step, it is further intended that there are compositions of the present invention that are essentially composed of or consist of the listed components, and processes and methods of the present invention that are essentially composed of or consist of the listed processing steps. Similarly, throughout this description, where a composition is described as being essentially composed of a particular component, or where a process and method is described as being essentially composed of a particular step, it is further intended that there are compositions of the present invention that are essentially composed of the listed components, and processes and methods of the present invention that are essentially composed of the listed processing steps.

[0233] In this application, when an element or component is said to be included in and / or selected from a list of enumerated elements or components, it should be understood that the element or component may be any of the enumerated elements or components, or may be selected from a group consisting of two or more of the enumerated elements or components.

[0234] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is referenced, unless otherwise understood from the context, that compound can be used in various embodiments of the compositions and / or methods of the invention. In other words, while embodiments in this application are described and depicted in such a manner that obvious and concise applications are described and depicted, it is intended and understood that embodiments can be combined or separated in various ways without departing from the teachings and the invention(s) of the invention. For example, it should be understood that all features described and depicted herein are applicable to all embodiments of the invention(s) described and depicted herein.

[0235] The expression "at least one of ~" should be understood to individually encompass each of the items listed after it, as well as various combinations of two or more of the listed items, unless otherwise understood from the context and usage. The expression "and / or" in relation to three or more listed items has the same meaning unless otherwise understood from the context.

[0236] As long as this method remains feasible, it should be understood that the order of the steps or the order in which specific actions are performed is not important. Furthermore, two or more steps or actions can be performed simultaneously.

[0237] Unless specifically requested, any use of examples or illustrative words herein, such as “such as” or “including,” is intended solely to better illustrate the invention and not to limit its scope. Language herein should not be construed as indicating any non-claiming element essential to the practice of the invention. [Examples]

[0238] The following examples are for illustrative purposes only and are not intended to limit the scope or content of the present invention in any way.

[0239] Example 1 This example describes the treatment of autoimmune diseases in subjects requiring treatment of autoimmune diseases by administering T cells genetically engineered to express CARs after administration of a preconditioning regimen (for example, a preconditioning regimen comprising one or more doses of cyclophosphamide, or essentially consisting thereof, but not comprising a dose of fludarabine).

[0240] T cells are collected from subjects with autoimmune diseases (e.g., autoimmune diseases selected from SLE, lupus nephritis, SLE with anti-dsDNA antibodies, pemphigus vulgaris (PV), mucosal PV, mucocutaneous PV, myasthenia gravis (MG), MuSK-associated MG, AChR MG, myositis, membranous nephropathy, antisynthetic enzyme syndrome, dermatomyositis, immune-mediated necrotizing myopathy, multiple sclerosis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, anti-NMDA receptor encephalitis, Lambert-Eaton syndrome, pemphigus foliaceus, acquired epidermolysis bullosa, bullous pemphigoid, Goodpasture syndrome, rheumatoid arthritis, systemic sclerosis, anti-human neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, immune thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, type 1 diabetes mellitus, Graves' disease, Hashimoto's disease, and Sjögren's syndrome). T cells are transduced using a vector encoding an anti-CD19 CAR, such as an anti-CD19 CAR containing the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3ζ intracellular domain, for example, a CAR containing the amino acid sequence of SEQ ID NO: 23. The transduced T cells are grown and optionally cryopreserved.

[0241] Prior to CAR T cell infusion, subjects are administered a preconditioning regimen containing, or essentially consisting of, one or more intravenous doses of cyclophosphamide. For example, subjects may be administered a preconditioning regimen such as those disclosed in Table 1. Control subjects or control groups, instead, receive a standard preconditioning dose, e.g., 1,000 mg / m². 2 The total dose of cyclophosphamide and 75-90 mg / m² 2 A total dose of fludarabine may be administered.

[0242] CAR T cell dose (e.g., 1 × 10⁻¹⁰) 6 ~1 × 10 7 The drug (individual cells / kg body weight) is administered intravenously to the target. After infusion, the target is monitored for B cell levels, white blood cell count, CAR T cell levels, and autoimmune disease markers (e.g., autoantibody levels).

[0243] The results of this example demonstrate that autologous CD19-CAR T cell therapy is effective in treating autoimmune diseases. This example also demonstrates that the use of a cyclophosphamide preconditioning regimen (e.g., the preconditioning regimen disclosed in Table 1) prior to CD19-CAR T cell therapy is effective in treating autoimmune diseases.

[0244] Example 2 This example describes a phase I / II, open-label, single-arm, randomized, parallel-group trial to investigate the safety and efficacy of autologous CD19-specific CAR T cells (CAB-001) in subjects with active systemic lupus erythematosus.

[0245] background SLE is a chronic autoimmune disorder characterized by autoantibody production and abnormal B-cell function. SLE presents with fluctuating severity and can cause tissue damage in various organs over time. Lupus nephritis (LN) is a common severe symptom of SLE and can lead to serious illness and death. There is no approved treatment for SLE, and available treatment options have limited efficacy in SLE patients and are associated with serious long-term side effects.

[0246] Investigational drug The investigational drug CAB-001 is an autologous chimeric antigen receptor (CAR) T cell that targets CD19. The CAR contains a fully human scFv that specifically binds to CD19, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The anti-CD19 scFv contains the amino acid sequence of SEQ ID NO: 9, and the construct contains the amino acid sequence of SEQ ID NO: 23.

[0247] Purpose and endpoint The primary objective of this study is to evaluate the safety and tolerability of CAB-001 over 28 days in subjects with active SLE, as measured by the incidence of adverse events (AEs) occurring within 28 days after CAB-001 injection, including dose-limiting toxicity (DLT) and CAB-001-related AEs.

[0248] The second objective of this study is to evaluate the safety and tolerability of CAB-001 over a 156-week period in subjects with active SLE, as measured by adverse events (AEs), vital signs, physical examination, and clinical laboratory tests occurring within 156 weeks after CAB-001 infusion.

[0249] A second additional objective of this study is to evaluate the effect of the CAB-001 regimen on the number of white blood cells containing B cells, as measured from baseline.

[0250] A second objective of this additional study is to evaluate the proliferation and persistence of CAB-001 in vivo, as measured by the approximate number of CAB-001 cells and CAB-001 labeling per cell number in the subjects.

[0251] A secondary objective of this additional study is to evaluate the effects of CAB-001 on SLE serum, as measured by changes from baseline in anti-double-stranded DNA (dsDNA) antibodies, complement component 3 (C3), complement component 4 (C4), and 50% hemolytic complement (CH50).

[0252] A secondary objective of this additional study is to evaluate the effect of CAB-001 on SLE disease activity, as measured by changes in urinary protein:creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), SLEDAI-2K score, Commonwealth Lupus Assessment Committee (BILAG)-2004 score, physician's general assessment (PGA) score, number of painful joints, cutaneous lupus erythematosus area and severity index (CLASI) score, and SLICC disability index (SDI); as well as the proportion of subjects achieving complete renal response (CRR), SRI-4, SRI-5, SRI-6, BICLA response, LLDAS, and DORIS remission criteria.

[0253] A second objective of this additional study is to evaluate the effect of CAB-001 on corticosteroid combinations and other combination SLE-related therapies, as measured by the change from baseline in doses of combination corticosteroids and other SLE-related therapies, the proportion of subjects achieving oral prednisone (or equivalent) doses of 5 mg / day or less, and the proportion of subjects who do not require SLE-related therapy.

[0254] A secondary objective of this additional study is to evaluate the effects of CAB-001 on patient-reported outcomes and health-related quality of life, as measured by changes from baseline on the 36-item shortened health questionnaire version 2 (SF-36v2), numerical pain rating scale (NRS), functional assessment of chronic disease treatment - fatigue (FACIT-F), patient-to-general assessment (PtGA), lupus quality of life scale (lupus QoL), and EQ-5D-5L score.

[0255] A secondary objective of this additional trial is to evaluate the effect of CAB-001 on disease relapse, as measured by the incidence of mild / moderate and severe disease relapses, as well as the time to mild / moderate and severe relapses, as defined according to the BILAG-2004 and modified SELENA-SLEDAI relapse index (SFI).

[0256] Additional objectives and endpoints relate to the sub-trials detailed in "Part B" below, which will be conducted to further evaluate the efficacy of CAB-001 in the treatment of lupus nephritis (LN).

[0257] The primary objective of this secondary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in achieving complete renal response at 52 weeks in subjects with lN, as measured by the proportion of subjects achieving complete renal response at 52 weeks.

[0258] The second objective of this secondary study is to compare the efficacy of CAB-001 and belimumab plus standard therapy in achieving complete renal response, as measured by the proportion of subjects achieving complete renal response at 24 weeks, the proportion of subjects achieving complete renal response at 24 and 52 weeks and not receiving other SLE-related therapies, the time to first achieving complete renal response, and the time to first achieving complete renal response and not receiving other SLE-related therapies.

[0259] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in UPCR response, as measured by the proportion of subjects achieving UPCR ≤ 0.5 mg / mg at 24 and 52 weeks, the time to first achievement of UPCR ≤ 0.5 mg / mg, and the change from baseline in UPCR at 24 and 52 weeks.

[0260] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in renal function, as measured by the change from baseline in estimated glomerular filtration rate (eGFR) at 24 and 52 weeks.

[0261] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in achieving SRI-4 response, as measured by the proportion of subjects achieving SRI-4 response at 24 and 52 weeks.

[0262] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in achieving BICLA response, as measured by the proportion of subjects achieving BICLA response at 24 and 52 weeks.

[0263] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in SLEDAI-2K response, as measured by the change from baseline SLEDAI-2K score up to week 52.

[0264] The second objective of this supplementary study is to compare the effectiveness of CAB-001 and belimumab plus standard treatment in achieving low disease activity (LLDAS), as measured by the proportion of subjects achieving Lupus low disease activity (LLDAS) at 24 and 52 weeks.

[0265] The second objective of this additional substudy is to compare the efficacy of CAB-001 and belimumab plus standard treatment in achieving remission, as measured by the proportion of subjects achieving definition of SLE remission (DORIS) at 24 and 52 weeks.

[0266] The second objective of this additional substudy is to compare the effects of CAB-001 and belimumab plus standard treatment on B cell count, as measured by the change in peripheral B cell count over time up to week 52.

[0267] The second objective of this additional substudy is to compare the effects of CAB-001 and belimumab plus standard treatment in SLE serum, as measured by the change from baseline in anti-dsDNA antibody levels up to week 52, and the change from baseline in C3, C4, and CH50 levels up to week 52.

[0268] A second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in corticosteroid use, measured as the change from baseline in concomitant corticosteroid dose at weeks 24 and 52, and as the proportion of subjects achieving oral prednisone or equivalent doses of 5 mg / day or less at weeks 24 and 52.

[0269] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in disease relapse, as measured by the incidence of mild / moderate and severe disease relapse up to week 52 according to BILAG-2000 and SFI, and by the time to mild / moderate and severe disease relapse.

[0270] The second objective of this supplementary study is to compare the effects of CAB-001 and belimumab plus standard treatment on patient-reported outcomes and health-related quality of life, as measured by changes from baseline in the SF-36v2 Health Questionnaire, Pain Numerical Rating Scale, FACIT-F, PtGA, Lupus QoL, and EQ-5D-5L scores at 24 and 52 weeks.

[0271] The second objective of this supplementary study is to evaluate the effect of CAB-001 on disease activity and response at weeks 56 to 156 after infusion, measured as changes from baseline in UPCR, eGFR, SLEDAI-2K, BILAG-2004, PGA, number of painful joints, CLASI, and SDI; proportion of subjects achieving complete renal response; proportion of subjects achieving complete renal response and not receiving other SLE-related therapies; proportion of subjects achieving UPCR ≤ 0.5 mg / mg; and proportion of subjects achieving SRI-4, SRI-5, SRI-6, and BICLA responses, as well as LLDAS and DORIS criteria.

[0272] The second objective of this supplementary study is to evaluate the effects of CAB-001 on steroid combination therapy and combination SLE-related therapies at weeks 56 to 156 after infusion, measured as changes in the dose of concomitant corticosteroids, the proportion of subjects achieving a dose of oral prednisone or equivalent of 5 mg / day or less, the proportion of subjects discontinuing prednisone use, changes in the dose of SLE-related therapies, and the proportion of subjects discontinuing all SLE-related therapies.

[0273] The second objective of this supplementary study is to evaluate the effects of CAB-001 on patient-reported outcomes and health-related quality of life at 56 to 156 weeks post-infusion, as measured by changes from baseline in the SF-36v2 Health Questionnaire, Pain Numerical Assessment Scale, FACIT-F, PtGA, Lupus QoL, and EQ-5D-5L scores.

[0274] The second objective of this additional substudy is to evaluate the effect of CAB-001 on SLE serum at 56 to 156 weeks post-infusion, as measured by changes from baseline in anti-dsDNA antibodies, as well as changes from baseline in C3, C4, and CH50.

[0275] The second objective of this supplementary study is to evaluate the safety and tolerability of CAB-001 in subjects with LN up to 156 weeks, as measured by AEs (including AESIs of particular interest), vital signs, physical examination, and clinical laboratory tests.

[0276] Additional objectives and endpoints relate to a second sub-trial, detailed in "Part B" below, which will be conducted to further evaluate the efficacy of CAB-001 in the treatment of non-renal SLE.

[0277] The primary objective of this secondary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment at 52 weeks in patients with non-renal SLE, as measured by the proportion of patients achieving complete SRI-4 response at 52 weeks.

[0278] The second objective of this secondary study is to compare the efficacy of CAB-001 and belimumab plus standard therapy in SRI-4 response, as measured by the proportion of patients achieving complete SRI-4 response at 24 weeks, the proportion of patients achieving complete SRI-4 response at 24 and 52 weeks, the time to first SRI-4 response, and the time to first SRI-4 response and being free from other SLE-related therapies.

[0279] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard therapy in achieving SRI-6 response, as measured by the proportion of subjects achieving SRI-6 response at 24 and 52 weeks, and the proportion of subjects achieving SRI-6 response at 24 and 52 weeks and not receiving other SLE-related treatments.

[0280] The second objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in achieving BICLA response, as measured by the proportion of subjects achieving BICLA response at 24 and 52 weeks.

[0281] The objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in SLE disease activity, as measured by the change from baseline in SLEDAI-2K score, BILAG-2004 score, PGA score, and the change from baseline in the number of affected joints at 24 and 52 weeks.

[0282] The objective of this additional substudy is to compare the efficacy of CAB-001 and belimumab plus standard treatment in terms of skin activity, as measured by the proportion of subjects achieving CLASI50 at 24 and 52 weeks.

[0283] The objective of this additional substudy is to compare the effectiveness of CAB-001 and belimumab plus standard treatment in achieving low disease activity (LLDAS), as measured by the proportion of subjects achieving Lupus low disease activity (LLDAS) at 24 and 52 weeks.

[0284] The objective of this additional substudy is to compare the efficacy of CAB-001 and belimumab plus standard treatment in achieving remission, as measured by the proportion of subjects achieving definition of SLE remission (DORIS) at 24 and 52 weeks.

[0285] The objective of this additional substudy is to compare the effects of CAB-001 and belimumab + standard treatment on B cell count, as measured by the change from baseline in peripheral B cell count over time up to week 52.

[0286] The objective of this additional substudy is to compare the efficacy of CAB-001 and belimumab plus standard treatment in SLE serum, as measured by the change from baseline in anti-dsDNA antibody levels up to week 52, and by the change from baseline in C3, C4, and CH50 levels up to week 52.

[0287] The objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab + standard treatment in corticosteroid use, measured as the change from baseline in concomitant corticosteroid dose at 24 and 52 weeks, and as the proportion of subjects achieving a low dose of oral prednisone or equivalent at 24 and 52 weeks.

[0288] The objective of this supplementary study is to compare the efficacy of CAB-001 and belimumab plus standard treatment in disease relapse, as measured by the incidence of mild / moderate and severe disease relapses up to week 52 according to the BILAG-2004 and modified SELENA-SLEDAI relapse index (SFI), and by the time to mild / moderate and severe relapse.

[0289] The second objective of this supplementary study is to compare the effects of CAB-001 and belimumab plus standard treatment on patient-reported outcomes and health-related quality of life, as measured by changes from baseline in SF-36v2, pain NRS, FACIT-F, PtGA, lupus QoL, and EQ-5D-5L scores at 24 and 52 weeks.

[0290] The objective of this additional substudy is to evaluate the effect of CAB-001 on disease activity at response times from weeks 56 to 156 after infusion, as measured by changes from baseline in UPCR, eGFR, SLEDAI-2K, BILAG-2004, PGA, number of painful joints, CLASI, and SDI, and also as the proportion of subjects achieving SRI-4, SRI-5, SRI-6, and BICLA responses, as well as LLDAS and DORIS criteria.

[0291] The second objective of this supplementary study is to evaluate the effects of CAB-001 on corticosteroid use and SLE-related treatments at weeks 56 to 156 after infusion, measured as changes in concomitant corticosteroid doses, the proportion of subjects achieving oral prednisone or equivalent doses of 5 mg / day or less, the proportion of subjects discontinuing prednisone use, changes in SLE-related treatment doses, and the proportion of subjects discontinuing all SLE-related treatments.

[0292] The second objective of this supplementary study is to evaluate the effects of CAB-001 on patient-reported outcomes and health-related quality of life at 56 to 156 weeks post-infusion, as measured by changes from baseline in the SF-36v2 Health Questionnaire, Pain Numerical Assessment Scale, FACIT-F, PtGA, Lupus QoL, and EQ-5D-5L scores.

[0293] The second objective of this additional substudy is to evaluate the effect of CAB-001 on SLE serum at 56 to 156 weeks post-infusion, as measured by changes from baseline in anti-dsDNA antibody levels, as well as changes from baseline in C3, CD4, and CH50 levels.

[0294] The second objective of this supplementary study is to evaluate the safety and tolerability of CAB-001 in subjects with LN up to 156 weeks, as measured by AEs (including AEs of particular interest), vital signs, physical examination, and clinical laboratory tests.

[0295] Test design This is a Phase 1 / 2 trial designed to evaluate the safety, tolerability, and efficacy of CAB-001 in adults with active SLE. The trial is in two parts. In Part A, single-arm CAB-001 treatment will be evaluated for safety and tolerability, and the dose to be used in Part B will be determined. In Part B, CAB-001 will be compared to belimumab in two independent sub-studies to evaluate its safety and efficacy in the treatment of patients with LN and patients with active SLE (non-renal SLE) who do not meet the criteria for LN. All subjects receiving CAB-001 will be followed for 156 weeks for efficacy and safety.

[0296] Part A: Open-label safety and tolerability testing The design of Part A of the study is summarized in Figure 2A. In Part A, a minimum of six subjects will be enrolled who have either LN (a minimum of three subjects) or SLE meeting the criteria for LN ("non-renal SLE"). All subjects will undergo leukocyte apheresis and the CAB-001 cell preparation will be manufactured. Following leukocyte apheresis and cell production, subjects will be administered the following standard preconditioning regimen: subjects will receive 25 mg / m² on days -5, -4, and -3. 2 The subjects received intravenous administration of fludarabine per day, with 1,000 mg / m² administered on day 3. 2 Cyclophosphamide is administered intravenously at a dose of 1 / day. On day 0, the subjects received 1 × 10⁶ doses. 6CAB-001 is infused at a dose of CAR T cells / kg body weight (A1 cohort). A minimum of 14 days must be observed between doses of any two subjects, and 28 days if dose-limiting toxicity (DLT) is observed. DLT is defined as any of the following adverse events directly related to CAB-001: (1) Cytokine release syndrome (CRS): occurring within 28 days of cell infusion and not improving to grade 1 or below within 7 days, grade 2 CRS according to the American Society for Transplantation and Cell Therapy (ASTCT); (2) Immune effector cell-associated neurotoxicity syndrome (ICANS): occurring within 28 days of cell infusion and not improving to grade 1 or below baseline within 7 days, grade 2 ICAN according to the ASTCT; (3) Clinically significant, not pre-existing, not due to disease progression, not related to other underlying conditions, and occurring within 28 days of cell infusion. (4) Grade 2 organ toxicity according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0, occurring within 7 days and not recovering to Grade 1 or below or to baseline (cardiac, cutaneous, gastrointestinal, hepatic, pulmonary, renal / genitourinary, or nervous system); (5) any Grade 3 or 4 CRS or any Grade 3 or 4 ICANs occurring within 28 days of cell infusion; or (6) Grade 3 or higher organ toxicity according to the CTCAE, occurring within 28 days of cell infusion, which is clinically significant, not pre-existing, not due to disease progression, and not related to any other underlying disease(s). If DLT is observed in two or more subjects, a lower dose of CAB-001 may be evaluated (e.g., Cohort A0; see Table 5).

[0297] [Table 5]

[0298] If one or fewer DLTs are observed within the cohort, the decision to proceed to Part B of the study will be based on translational study data of the biological response at day 28, including B cell depletion. If incomplete B cell depletion is observed in zero subjects at day 28, the study may proceed to Part B without changing the CAR T cell dose. If incomplete B cell depletion is observed in one subject at day 28, the CAR T cell dose may be increased, or the study may proceed to Part B at the current dose (e.g., cohorts A2 and A3, as shown in Table 5). If incomplete B cell depletion is observed in two subjects at day 28, the dose may be increased.

[0299] All participants in Part A will be evaluated over 156 weeks for safety, tolerability, efficacy, and quality of life in a manner suitable for assessing the research objectives.

[0300] Part B: Sub-study of Activated Control Agent Two independent subtrials will be conducted to further evaluate the efficacy of CAB-001 compared to belimumab (an approved treatment for both LN and non-renal SLE) in addition to standard therapy. Eligible subjects meeting the diagnostic criteria for LN will be enrolled in the LN subtrial, while other eligible subjects will be enrolled in the non-renal SLE subtrial. Approximately 66 subjects will be enrolled in the LN subtrial and approximately 90 subjects in the non-renal SLE subtrial, for a total of approximately 156 subjects. The design of Part B of the trial is summarized in Figures 2B-2C.

[0301] Participants in each secondary trial will be randomized in a 2:1 ratio to one of two groups: Group B1 (CAB-001) and Group B2 (belimumab). Participants in Group B1 will undergo leukocyte apheresis to produce CAB-001 CAR T cells. Group B1 will receive 25 mg / m² on days -5, -4, and -3. 2 Fludarabine per day, and 1,000 mg / m² on day 3. 2 Group B1 receives a complete preconditioning regimen of cyclophosphamide (see Table 6) intravenously on day 0, the dose selected in Part A (3 × 10⁴). 5 ~1 × 107 CAR T cells / kg, e.g., 3 × 10⁶ 5 , 1 x 10 6 , 3x10 6 , or 1 × 10 7 CAB-001 is administered intravenously at a dose of (100 CAR T cells / kg). Participants will be evaluated for safety, tolerability, efficacy, and quality of life at regular check-ups up to week 52. After the 52-week check-up, participants will be followed up and evaluated as needed until week 156.

[0302] Participants in Group B2 will undergo leukocyte apheresis to produce CAB-001 CAR T cells. Participants will receive standard doses of belimumab intravenously for 12 months in conjunction with standard treatment. Safety, tolerability, efficacy, and quality of life will be evaluated as appropriate during routine visits up to week 52. At the end of the week 52 visit, eligible Group B2 participants will be permitted to receive CAB-001 ("crossover participants"). Depending on newly obtained data from Part A, crossover participants in Group B2 will receive either the complete preconditioning regimen used in Group B2, or, instead of the complete preconditioning regimen, 12.5 mg / m² on days -5, -4, and -3. 2 Fludarabine per day, and 500 mg / m² on day 3. 2 Participants in the crossover group B2 will receive a reduced preconditioning regimen of cyclophosphamide per day. Crossover participants will then receive CAB-001 at the dose used in group B1. Participants will be evaluated for safety, tolerability, efficacy, and quality of life over a total of 156 weeks after infusion.

[0303] [Table 6]

[0304] Exam period For subjects in Group B1 of Parts A and B, the pre-treatment period, including screening, leukocyte apheresis (cell collection), cell production, preconditioning, and pre-infusion (baseline) evaluation, lasts approximately 4–18 weeks. CAB-001 infusion lasts approximately 1 day. The post-treatment period lasts 156 weeks and includes follow-up visits for evaluations necessary for the study.

[0305] For subjects in Group B2 of Part B, the pre-treatment period, including screening and leukocyte apheresis (cell collection), lasts up to 14 weeks. Treatment with belimumab lasts approximately 52 weeks. For crossover subjects, this is followed by preconditioning, treatment with CAB-001, and post-infusion follow-up, which lasts up to approximately 162 weeks.

[0306] After the completion of the post-treatment period of up to 156 weeks, all subjects who received CAB-001 will be followed in a long-term follow-up (LTFU) period. During the LTFU period, subjects will be followed for up to 15 years after CAB-001 injection (excluding any pre-injection period and belimumab treatment).

[0307] Inclusion / Exclusion Criteria The inclusion criteria for Part A of the study are as follows: (1) Ability to submit informed consent; (2) Minimum age 18 and maximum age 65; (3) Performance status of 0-1 in the East Coast Cancer Group (ECOG); (4) Clinical diagnosis of SLE based on the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) adult SLE classification criteria; (5) Positive antinuclear antibody (ANA) titer of 1:80 or higher or positive for anti-dsDNA antibody at screening; (6) For LN subjects, regardless of the presence or absence of Class V, the 2003 ISN / RPS criteria were used. (7) Having active class III or IV lupus nephritis as confirmed by biopsy (biopsy must be performed within 6 months prior to the screening visit or during the screening period); (8) Having been diagnosed with active SLE as defined by the following criteria: a. LN eligibility: UPCR ≥ 1 mg / mg in two morning first-urine samples during screening, despite prior or current treatment with standard therapy including corticosteroids and either mycophenolate mofetil (MMF) / mycophenolate (MPA) or cyclophosphamide, or b.Non-renal SLE patients: SLEDAI-2K ≥ 8 at screening and clinical SLEDAI-2K ≥ 6 (excluding headache, alopecia, mucosal ulcers, fever, and organic brain syndrome) despite prior or current treatment with standard therapy including corticosteroids, rituximab or other B-cell depletion agents, cyclophosphamide, MMF / MPA, azathioprine, methotrexate, 6-mercaptopurine, sirolimus, tacrolimus, thalidomide, leflunomide, mizoribine, aniflorumab, and belimumab; (8a) If currently receiving standard immunosuppressive therapy (including MMF / MPA, oral cyclophosphamide, azathioprine, antimalarial therapy, methotrexate, 6-mercaptopurine, sirolimus, tacrolimus, thalidomide, leflunomide, or mizoribine), that treatment is, (8) If currently receiving a renin-angiotensin-aldosterone system inhibitor (including direct renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and mineralocorticoid receptor blockers), the dose must be stable for at least two weeks prior to screening; (8) If currently receiving oral corticosteroids, the dose must be stable at 30 mg or less of prednisone or an equivalent for at least two weeks prior to screening; (9) Estimated glomerular filtration rate of 30 mL / min / 1.73 m using the equation of the chronic kidney disease epidemiological collaborative study. 2(10) Having sufficient liver function as evidenced by an alanine aminotransferase (ALT) value ≤ 2.5 × ULN; (11) In the judgment of the investigator, the subject requires additional systemic therapy for SLE and is a suitable candidate for treatment with CAB-001; and (12) Women of childbearing potential (WOCBP) must agree to use contraception from screening until at least 52 weeks after CAB-001 infusion (acceptable contraception methods include intrauterine devices, hormonal contraception, complete sexual abstinence, vasectomy of the partner, or monogamy with a male partner with azoospermia).

[0308] The exclusion criteria for Part A of the study are as follows: (1) Treatment with rituximab or other B-cell depletion agents within 6 months prior to screening, or within 3 months if there is a test result indicating the presence of CD19+ B cells; (2) Treatment with voclosporine or other calcineurin inhibitors within 2 months prior to screening; (3) Treatment with aniflorumab or other biological agents within 3 months prior to screening; (4) For LN subjects only, if sclerosis is observed in more than 50% of the glomeruli on renal biopsy; (5) Diagnosis of cancer other than basal cell carcinoma or squamous cell carcinoma of the skin or cervical intraepithelial carcinoma that has been excised and cured and at least 5 years have passed since excision; (6) Major surgery (including joint surgery) scheduled within 52 weeks after CAB-001 injection; (7) Contraindication of leukocyte apheresis. (8) A history of anaphylactic or severe systemic reaction to fludarabine, cyclophosphamide, or any of their metabolites; (9) An absolute lymphocyte count <1,000 / μL at screening; (10) Positive results for human immunodeficiency virus (HIV), hepatitis C antibody, or hepatitis B surface antigen at screening, or evidence of active or chronic tuberculosis (TB); (11) An active infection requiring medical intervention at screening; (12) An autoimmune disease other than SLE requiring immunosuppressive therapy; (13) A kidney disease other than active lupus nephritis; (14) A serious, progressive, or uncontrolled kidney, liver, hematological, or gastrointestinal disease, including a serious and uncontrolled infection, e.g., sepsis and opportunistic infection. (15) If the patient currently has symptoms of lung disease, mental illness, heart disease, neurological disease, or brain disease; (16) If, in the judgment of the investigator, participation in this study could expose the patient to an unacceptable risk or a complication that could interfere with the evaluation of the efficacy or safety of the investigational drug or the study procedure; (17) A history of CAR T-cell therapy; (18) A history of solid organ (heart, liver, kidney, lung) transplantation or hematopoietic stem cell transplantation; (19) A history of live vaccine administration within 30 days prior to the infusion visit; (10) A pregnant or breastfeeding woman, or a woman planning to become pregnant within 52 weeks after CAB-001 infusion;(20) If the protocol cannot be followed or is unwilling to follow it;

[0309] The inclusion criteria for Part B of the trial are as follows: (1) Ability to submit informed consent; (2) Minimum age 18 and maximum age 65; (3) Performance status of 0-1 in the East Coast Cancer Group (ECOG); (4) Clinical diagnosis of SLE based on the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) adult SLE classification criteria; (5) ANA positive with a ratio of 1:80 or higher or positive for anti-dsDNA antibodies; (6) For LN subjects, whether or not they have Class V using the 2003 ISN / RPS criteria. (7) The subject has been diagnosed with active lupus nephritis of class III or IV as confirmed by biopsy (biopsy must be performed within 6 months prior to the screening visit or during the screening period); (8) The subject has been diagnosed with active SLE and further has LN or non-renal SLE and meets the following criteria: a. LN supplementary test: UPCR ≥ 1 mg / mg in two morning first-urine samples during screening despite treatment with standard therapy using glucocorticoids and either MMF / MPA or cyclophosphamide, or b.Non-renal SLE subtrial: SLEDAI-2K ≥ 8 at screening and clinical SLEDAI-2K ≥ 6 (excluding headache, alopecia, mucosal ulcers, fever, and organic brain syndrome) despite prior or current treatment with standard therapy including corticosteroids, rituximab or other B-cell depletion agents, cyclophosphamide, MMF / MPA, azathioprine, 6-mercaptopurine, methotrexate, sirolimus, tacrolimus, thalidomide, leflunomide, mizoribine, and aniflorumab; (8a) If receiving standard immunosuppressive therapy (including MMF / MPA, oral cyclophosphamide, azathioprine, 6-mercaptopurine, antimalarial drugs, methotrexate, sirolimus, tacrolimus, thalidomide, leflunomide, or mizoribine), (8b) If receiving a renin-angiotensin-aldosterone system inhibitor (including direct renin inhibitors, ACE inhibitors, ARBs, and mineralocorticoid receptor blockers), the subject must have a stable dose for at least two weeks prior to screening; (8c) If receiving an oral corticosteroid, the subject must be receiving 30 mg or less of prednisone or an equivalent, and the dose must have been stable for at least two weeks prior to screening; (9) The estimated glomerular filtration rate using the equation of the chronic kidney disease epidemiological collaborative study is 30 mL / min / 1.73 m. 2 (10) Having sufficient liver function as evidenced by an ALT value ≤ 2.5 × ULN; (11) In the judgment of the investigator, the subject requires additional systemic therapy for SLE and is a suitable candidate for treatment with CAB-001; and (12) WOCBP must agree to use contraception from screening until at least 52 weeks after CAB-001 infusion or, if applicable, 16 weeks after the last belimumab infusion (acceptable contraceptions include intrauterine devices, hormonal contraception, complete sexual abstinence, vasectomy of the partner, or monogamy with a male partner with azoospermia).

[0310] The exclusion criteria for Part B of the trial are as follows: (1) Treatment with rituximab or other B-cell depletion agents within 6 months prior to screening, or within 3 months if there is a test result indicating the presence of CD19+ B cells; (2) Prior treatment with belimumab; (3) Treatment with voclosporine or other calcineurin inhibitors within 2 months prior to screening; (4) Treatment with aniflorumab or other biological agents within 3 months prior to screening; (5) For the LN subsequent trial, if sclerosis is observed in more than 50% of glomeruli on renal biopsy; (6) Diagnosis of cancer other than basal cell carcinoma or squamous cell carcinoma of the skin or cervical intraepithelial carcinoma that has been excised and cured and at least 5 years have passed since excision; (7) Major surgery (including joint surgery) scheduled within 52 weeks after CAB-001 infusion or the first belimumab infusion; (8) Contraindication of leukocytosis apheresis; (9) L For the N sub-trial, a history of intolerance, anaphylactic reaction, or severe systemic reaction to MMF / MPA; (10) a history of anaphylactic reaction or severe systemic reaction to fludarabine, cyclophosphamide, or any of their metabolites; (11) absolute lymphocyte count <1,000 / μL at screening; (12) positive for HIV, hepatitis C antibody, or hepatitis B surface antigen at screening, or evidence of active or chronic TB; (13) having an active infection requiring medical intervention at screening; (14) having an autoimmune disease other than SLE requiring immunosuppressive therapy; (15) having a renal disease other than active lupus nephritis; (16) a serious, progressive, or uncontrolled renal, hepatic, hematological, or gastrointestinal disease, including a serious and uncontrolled infection, e.g., sepsis and opportunistic infection. (17) If the patient currently has symptoms of lung disease, mental illness, heart disease, neurological disease, or brain disease; (18) If the patient has a comorbidity that, in the judgment of the investigator, could expose the patient to an unacceptable risk by participating in this study, or that could interfere with the evaluation of the efficacy or safety of the investigational drug or the study procedure; (19) A history of CAR T-cell therapy; (20) A previous solid organ (heart, liver, kidney, lung) transplant or hematopoietic stem cell transplant; (21) Having received a live vaccine within 30 days of the pre-infusion visit;(21) If a woman is pregnant or breastfeeding, or plans to become pregnant within 52 weeks after CAB-001 injection or the first belimumab injection; or (22) If she is unable or unwilling to comply with the protocol.

[0311] Example 3 This example describes a Phase 1 / 2 open-label trial designed to evaluate the safety and efficacy of CAB-001 in participants with generalized myasthenia gravis.

[0312] background Myasthenia gravis (MG) is a rare autoimmune disease characterized by an autoantibody reaction that causes incomplete signal transmission at the neuromuscular junction, resulting in a distinctive pattern of muscle weakness. In about 15% of MG patients, symptoms are limited to the ocular muscles and are called ocular MG (Gilhus et al. (2019) Nat. Rev. Dis. Primers 5(1):30). Patients with generalized MG usually exhibit muscle weakness in numerous other voluntary muscle groups, including the limb muscles, bulbar muscles, and respiratory muscles, in addition to symptoms associated with eye disease (Gilhus et al. (ibid.)). Severe muscle weakness due to MG can have a devastating impact on a patient's quality of life, including dysarthria, dysphagia, visual impairment, shortness of breath, motor impairment, and myasthenic crisis, which is a pulmonary failure episode requiring mechanical ventilation (Engel-Nitz et al., (2018) Muscle Nerve 1(1):16-22, Wendell and Levine (2011) Neurohospitalist 1(1):16-22).

[0313] Myosopharyngeal malformation (MG) is considered a typical example of a B-cell-mediated autoimmune disease in which the majority of patients have autoantibodies directed against molecular elements of the neuromuscular junction, leading to disruption of receptor signaling and potentially complement-mediated tissue damage (Yi et al., (2018) Muscle Nerve 57(2):172-184). In approximately 85% of patients, these autoantibodies are directed against muscle acetylcholine receptors (AChRs). Less frequently identified autoantibodies target muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein receptor-associated protein 4 (LRP4), respectively, in 6% and 2% of MG patients (Dresser et al., (2021) J.Clin.Med.10(11):2235). Currently, there is no treatment for MG. Available treatment options have limited efficacy, focus only on specific symptoms, and are associated with serious long-term side effects.

[0314] Investigational drug The investigational drug CAB-001 is an autologous chimeric antigen receptor (CAR) T cell that targets CD19. The CAR contains a fully human scFv that specifically binds to CD19, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The anti-CD19 scFv contains the amino acid sequence of SEQ ID NO: 9, and the construct contains the amino acid sequence of SEQ ID NO: 23 (or SEQ ID NO: 27, which contains the CD8α signal peptide).

[0315] Purpose and endpoint The primary objective of this study is to evaluate the safety and tolerability of CAB-001 in participants with generalized myasthenia gravis, as measured by the incidence of adverse events (AEs) occurring within 28 days after CAB-001 injection.

[0316] The additional purpose of this study is to evaluate the safety and tolerability of CAB-001 in participants with generalized MG over a 156-week period, as measured by adverse events (AEs), vital signs, physical examination findings, and laboratory results occurring within 156 weeks after CAB-001 infusion.

[0317] The additional objective of this study is to evaluate the effects of CAB-001 on hematological and immunological parameters, including WBC, T, B, NK, and myeloid populations, as measured by changes from baseline in fractionated WBC and T, B, and NK cell counts, as well as immunophenotypic subpopulations, after CAB-001 infusion.

[0318] The additional objective of this study is to evaluate the persistence and pharmacokinetics of CAB-001 after infusion, as measured by the number and percentage of CAB-001-positive cells in the peripheral blood of participants over time.

[0319] An additional objective of this study is to evaluate the effect of CAB-001 on MG serum, as measured by the change from baseline in autoantibody levels and / or status in participants with MG-specific autoantibodies (e.g., anti-AChR, anti-MuSK, anti-LRP4).

[0320] The additional objective of this study is to evaluate the effect of CAB-001 on systemic myasthenia gravis disease activity, measured as changes from baseline in MG-ADL, QMG, MGC, MGFA-PIS, and MGII, as well as the proportion of participants achieving MSE (MG-ADL score of 0 or 1), MG-ADL response (improvement of ≥2.0 points from baseline), QMG response (improvement of ≥3.0 points from baseline), and MGC response (improvement of ≥3.0 points from baseline).

[0321] The additional objective of this trial is to evaluate the time to achieve disease response after CAB-001 administration, as measured by the time to achieve MSE for MG-ADL response, QMG response, and MGC response.

[0322] The additional objective of this trial is to evaluate the effects of CAB-001 on patient-reported outcomes, measured as changes in MG-QOL 15r, Neuro-QoL fatigue, and / or EQ-5D-5L.

[0323] The additional objectives of this trial are to evaluate the effect of CAB-001 on disease relapse, as measured by (1) the incidence of clinical exacerbation, including the use of rescue treatment and MG crises; (2) the incidence of hospitalization and MG-related hospitalization, including days of hospitalization, days in the ICU, and days of ventilatory support; and (3) the effect of CAB-001 on disease relapse, as measured by time to clinical exacerbation.

[0324] An additional objective of this trial is to evaluate the effect of CAB-001 on drug-free response, as measured by the proportion of participants achieving minimal symptom onset, MG-ADL response, QMG response, and / or MGC response.

[0325] An additional objective of this trial is to evaluate the time to achieve drug-free response after CAB-001 infusion, as measured by the time to achieve minimal symptom onset without medication, MG-ADL response, QMG response, and / or MGC response.

[0326] Test design The design of this study is summarized in Figure 3. This study will enroll participants with generalized MG diagnosed as class II, III, or IV by the Myasthenia Gravis Foundation (MGFA). Participants will be tested in two cohorts based on the autoantibody status identified in MG: (1) an acetylcholine receptor (AChR) antibody-positive cohort, including participants with autoantibodies directed against AChR based on medical records or screening tests; and (2) an AChR antibody-negative cohort, including participants with negative results for autoantibodies directed against AChR based on medical records or screening tests. For cohort (2), participants must have autoantibodies directed against muscle-specific tyrosine kinase (MuSK) or low-density lipoprotein receptor-associated protein 4 (LRP4), or have seronegative MG, based on medical records or screening tests.

[0327] Each participant undergoes leukocyte apheresis, and a CAB-001 cell preparation is manufactured for each participant. In summary, autologous T cells are genetically modified ex vivo by transduction using a lentiviral vector. Fresh leukocyte apheresis products are collected and cryopreserved in several aliquots. One frozen aliquot is used for production in one manufacturing cycle. After the manufacturing process is complete, the drug product is formulated and cryopreserved.

[0328] Following leukocyte apheresis and cell production, participants will be administered a preconditioning regimen. The preconditioning regimen will be a standard preconditioning regimen (e.g., 25 mg / m² on days -5, -4, and -3). 2 Fludarabine is administered intravenously, and 1,000 mg / m² is given on day 3. 2 This may be cyclophosphamide administered intravenously, or it may be a preconditioning regimen consisting solely of cyclophosphamide (as described in Table 1, for example). CAB-001 is administered to participants by intravenous infusion in one of the following four doses: 1 × 10⁻¹⁶ 6 Individual cells / kg, 3×10 5 Individual cells / kg, 3×10 6 Individual cells / kg, 1×10 7 Individual cells / kg.

[0329] Participants will be evaluated for CAB-001-related toxicity and its effects on their underlying conditions during the initial 28-day evaluation period. Participants will be monitored on the day of infusion, with subsequent visits approximately 5, 8, 15, 22, and 29 days after infusion. In addition to routine chemical and hematological tests, clinical laboratory tests for PK sampling, coagulation panels, CRS lab tests, serum cytokine assessments, and CAR T assessments will be performed up to day 29. After the evaluation on day 29 after infusion, participants will be evaluated approximately once every 4 weeks (±7 days) in the first year, approximately every 12 weeks (±14 days) in the second year, and approximately every 26 weeks (±30 days) in the third year. All participants will be evaluated for safety, tolerability, efficacy, and quality of life for 156 weeks after infusion. Participants may also be monitored as needed for 15 years after treatment.

[0330] Inclusion / Exclusion Criteria The inclusion criteria for participation in this trial include: (1) being able to provide informed consent; (2) being between 18 and 70 years of age; (3) having MG with generalized muscle weakness that meets the criteria defined by MGFA class II, III, IVa, and IVb, and the diagnosis being supported by meeting at least one of the following two criteria: a. positive serological test for anti-AChR, anti-MuSK, or anti-LRP4 antibody based on medical history records or screening tests, or b. evidence of therapeutic response to acetylcholinesterase inhibitors and a history of neuromuscular abnormalities as shown by repetitive nerve stimulation or single-fiber electromyography (wherein seronegative cases, all must be negative for congenital myasthenic syndrome genetic testing, and in cases with only jitter abnormalities, mitochondrial disease must be excluded); (4) cholinesterase inhibitors, corticosterone (d) The total score of myasthenia gravis activities of daily living (MG-ADL) at screening is ≥6 despite a history of at least 12 weeks of standard treatment with at least two prior therapies, including nonsteroidal immunosuppressants, B-cell depletion therapy (e.g., rituximab), FcRn inhibitors (e.g., fgaltigimod, rozanolixizumab), complement inhibitors (e.g., eculizumab, ravulizumab, zircoplan), IVIg or subcutaneous immunoglobulin therapy, immunoadsorption, or plasmapheresis, or current standard treatment; (5) If the patient is currently receiving standard treatment for MG, the treatment must be at a stable dose prior to screening, except for dose reductions due to safety or tolerability, as follows: a. Acetylcholinesterase inhibitors: stable dose for at least 2 weeks; b. Corticosteroids: prednisone or equivalent of 30 mg or less and stable dose for at least 2 weeks; c.Nonsteroidal immunosuppressants (e.g., azathioprine, methotrexate, cyclosporine, tacrolimus, mycophenolate mofetil / mycophenolate, CY): initiated at least 12 weeks prior to screening and dose stable for at least 4 weeks); (6) For immunocompromised individuals, all currently recommended vaccinations, including COVID-19 / Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), must be received prior to or during screening in accordance with U.S. Centers for Disease Control and Prevention (CDC) or facility guidelines (live vaccines must be administered at least 30 days prior to pre-infusion visit, and non-live vaccines must be administered to participants at least 2 weeks prior to the start of investigational drug infusion, and if possible, non-live vaccines should be administered to leukocytes (7) Clinical stability as assessed by vital signs at screening, including a. systolic blood pressure ≥90 mmHg and ≤170 mmHg, b. diastolic blood pressure ≥55 mmHg and ≤105 mmHg, c. pulse rate ≥50 and ≤110 beats / min, d. respiratory rate ≥12 and ≤20 breaths / min, and e. no fever; (8) Reproductively capable women must agree to use two acceptable methods of contraception from screening until at least 52 weeks after CAB-001 injection; and (9) Reproductively capable, sexually active men receiving CAB-001, including vasectomized men (who have not been confirmed infertile by clinical testing), must use spermicide-treated condoms from screening until at least 52 weeks after CAB-001 injection.

[0331] Exclusion criteria include patients with any of the following criteria: (1) Class I or V MGFA; (2) Patients deemed unsuitable for participation in the study by the investigator (e.g., those at risk of requiring intubation during the study); (3) A history of active or untreated thymoma, thymic carcinoma, or thymic malignancy; (4) A history of thymectomy within 52 weeks of screening; (5) Clinical evidence of other serious medical comorbidities, complications, or worsening of drug-induced muscle weakness, or recent major surgery or planned major surgery that could interfere with the study results or pose an excessive risk to the participant; (6) Use of any of the following treatments within the specified period: a. Eculizumab within 12 weeks prior to screening, Zircoplan within 5 weeks prior to screening, or ravulizumab within 40 weeks prior to screening (however, complement test results at screening (i.e., total hemolytic complement [ (1) If the CH50 measurement is normal, (2) b. Rituximab or other B-cell depletion agent within 26 weeks prior to screening, or within 12 weeks prior to screening if there is a test result indicating the presence of CD19+ B cells at the time of screening, and c. Any investigational drug within 4 weeks or 5 half-lives (whichever is longer); (7) Contraindications to leukocyte apheresis; (8) History of anaphylactic or severe systemic reaction to fludarabine, cyclophosphamide, or any of their metabolites; (9) If the human immunodeficiency virus (HIV), hepatitis C virus (HCV) antibody, or hepatitis B surface antigen test is positive at screening, or if there is evidence of active or chronic tuberculosis; (10) Any of the following clinical laboratory values ​​at screening: a. Absolute lymphocyte count <500 / μL, b. Absolute neutrophil count <1,000 / μL, c. Hemoglobin <8 g / dL, d.(11) Active infection requiring medical intervention at the time of screening; (12) Active inflammatory autoimmune disorder requiring immunosuppressive therapy other than MG, or other neurological or neuromuscular disorders that interfere with accurate assessment of clinical symptoms; (13) Known malignancy or history of malignancy, excluding focal cutaneous basal cell carcinoma, focal cutaneous squamous cell carcinoma, or cervical intraepithelial neoplasia, with no evidence of recurrence for more than 3 years prior to screening; (14) Severe, progressive, or uncontrolled kidney, liver, hematological, or gastrointestinal disease, including severe and uncontrolled infections such as sepsis and opportunistic infections. , if symptoms of lung disease, mental illness, heart disease, associated neurological disorder, or brain disease are currently present; (15) Chronic lung disease, including: a. Resting oxygen saturation <90% without oxygen supplementation, b. Forced vital capacity (FVC) <60% if measured; (16) Cardiac dysfunction or clinically significant heart disease, including: a. Unstable angina or myocardial infarction or coronary artery bypass graft within 6 months prior to leukocytosis, b. Stage III or IV congestive heart failure by the New York Heart Association, c. Clinically significant cardiac arrhythmia (e.g.) (b) a history of ventricular tachycardia, complete left bundle branch block, or high-grade atrioventricular block; (d) a history of severe non-ischemic cardiomyopathy; and (e) left ventricular ejection fraction <45% as assessed by echocardiography or multi-gate acquisition scan (if performed) within 8 weeks of leukocytosis; (17) prior treatment with genetically modified T-cell therapy with permanent genetic modification; (18) prior solid organ (heart, liver, kidney, lung) transplantation or hematopoietic stem cell transplantation; (19) pregnant or lactating women; (20) unwillingness or inability to comply with the protocol.

[0332] The combination of CD19-CAR T-cell therapy and a preconditioning regimen is expected to be effective in treating myasthenia gravis.

[0333] Example 4 This example describes a Phase 1 / 2 open-label trial designed to evaluate the safety and efficacy of CAB-001 in adult participants with systemic sclerosis.

[0334] background Systemic sclerosis (SSc) is a rare, heterogeneous, potentially fatal, multisystem chronic autoimmune disorder. It is characterized by progressive cutaneous and visceral fibrosis, diffuse fibroproliferative angiopathy, and autoimmunity (Truchetet et al. (2023) Clin. Rev. Allergy Immunol. 64(3):262-283). The clinical presentation and course of SSc in individual patients are highly heterogeneous, and life expectancy is variable, mainly influenced by the extent of pulmonary and cardiac lesions.

[0335] SSc patients are classified into focal cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc; also known as diffuse systemic sclerosis) based on the severity of their skin lesions. Approximately one-third of patients are diagnosed with dcSSc, the more severe form of SSc (Jaafar et al. (2021) Arthrisis Res.Ther.23(1):170). In lcSSc, skin fibrosis is limited to the fingers (sclerodactyly), distal limbs, and face, while in dcSSc, the trunk and proximal limbs are also affected. In lcSSc patients, Raynaud's phenomenon usually precedes the skin lesions and other disease symptoms by several months to several years (Allanore et al. (2015) Nat.Rev.Dis.Primers 1:15002). Patients with dcSSc have a poorer prognosis, exhibiting widespread skin changes that can affect any area of ​​the body and rapid disease progression. Progression from Raynaud's phenomenon to skin thickening can occur within one year, and visceral vascular damage and fibrosis, including the gastrointestinal tract, heart, lungs, and kidneys, can occur within five years of diagnosis. Patients with dcSSc have an annual disease-related mortality rate of 5% to 12% (Allanore (ibid.), Milanetti et al. (2011) Curr. Stem Cell. Res. Ther. 6(1):16-28).

[0336] The involvement of pro-inflammatory cytokines (e.g., interleukin [IL]6, IL-13, and tumor necrosis factor [TNF]) and transforming growth factor β, as well as the presence of autoantibodies, are useful in both the diagnosis and classification of SSc. Antinuclear antibodies (ANA) are positive in approximately 90% of SSc cases. Anti-centromere antibodies and anti-Th / To antibodies are more commonly found in lcSSc than in dcSSc. Anti-topoisomerase I antibodies and anti-ribonucleic acid (RNA) polymerase III antibodies are more frequently found in dcSSc (Allanore et al. (ibid.), Cavazzana et al. (2023) Clin. Rev. Allergy Immunol. 64(3):412-430, Truchetet (ibid.), Yang et al. (2020) Front. Med. (Lausanne) 7:587773). Anti-U3-RNP (fibrillarin) antibodies are associated with a distinct clinical phenotype and an overall poor prognosis (male patients, African-Caribbean ancestry, young age at diagnosis, higher risk of visceral lesions such as pulmonary hypertension [PAH] and gastrointestinal and cardiac lesions) (Cavazzana (previously cited)).

[0337] Interstitial lung disease (ILD) and pulmonary hypertension together account for approximately 50% of deaths, followed by renal and cardiac complications (Cottin and Brown (2019) Respir.Res.20(1):13, Winstone et al. (2014) Chest 146(2):422-436). When disease progression is most rapid, mortality is highest in the first five years, with cumulative survival rates at 5 and 10 years being 70% and 50%, respectively. Esophageal symptoms are the second most frequently reported SSc findings (after cutaneous), with 50%–90% of patients reporting symptoms of dysphagia, heartburn, or gastroesophageal reflux disease (Abu-Shakra et al. (1994) Arthritis Rheum.24(1):29-39, Sjogren (1994) Arthritis Rheum.37(9):1265-1282). Furthermore, some patients show objective evidence of esophageal motility disorders through functional lumen imaging probe (FLIP) panometry, which provides a comprehensive assessment of esophageal function, distensibility, and contractility during endoscopy (Carlson et al. (2015) Curr.Rheumatol.Rep.17(1):475).

[0338] Currently, there is no cure for SSc, and no FDA-approved pharmaceutical products for treating SSc. Current treatment options are primarily limited to immunosuppressants and targeted biological agents, used to minimize the impact of the disease and the development of major complications (Kowal-Bielecka et al. (2017) Ann.Rheum.Dis.76(8):1327-1339). These treatments have little effect on delaying disease progression, and the prognosis for SSc patients, especially dcSSc patients, remains poor (Elhai et al. (2012) Rheumatology (Oxford) 51(6):1017-1026, Sullivan et al. (2018) N.Engl.J.Med.378(1):35-47). Current treatments aim to control symptoms, prevent imminent clinical outcomes, reduce organ damage, and reduce long-term disease status and mortality.

[0339] Investigational drug The investigational drug CAB-001 is an autologous chimeric antigen receptor (CAR) T cell that targets CD19. The CAR contains a fully human scFv that specifically binds to CD19, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The anti-CD19 scFv contains the amino acid sequence of SEQ ID NO: 9, and the construct contains the amino acid sequence of SEQ ID NO: 23 (or SEQ ID NO: 27, which contains the CD8α signal peptide).

[0340] Purpose and endpoint The primary objective of this study is to evaluate the safety and tolerability of CAB-001 in subjects with systemic sclerosis at risk of organ disease progression, as measured by the incidence of adverse events (AEs) occurring within 28 days after CAB-001 injection.

[0341] The additional purpose of this study is to evaluate the safety and tolerability of CAB-001 in subjects with systemic sclerosis over a 156-week period, as measured by adverse events (AEs), vital signs, physical examination findings, and laboratory results occurring within 156 weeks after CAB-001 infusion.

[0342] The additional objective of this study is to evaluate the effects of CAB-001 on hematological and immunological parameters, including WBC, T, B, NK, and myeloid populations, as measured by changes from baseline in fractionated WBC and T, B, and NK cell counts, as well as immunophenotypic subpopulations, after CAB-001 infusion.

[0343] The additional objective of this study is to evaluate the persistence and pharmacokinetics of CAB-001 after infusion, as measured by the number and percentage of CAB-001-positive cells in the peripheral blood of participants over time.

[0344] The additional objectives of this trial are: (1) the proportion of subjects achieving the revised CRISS criteria at 12, 24, 52, 80, 104, and 156 weeks after CAB-001 infusion; (2) the proportion of subjects achieving the revised CRISS criteria without immunosuppressive therapy at 12, 24, 36, 52, 80, 104, and 156 weeks after CAB-001 infusion; and (3) the response to each individual element of the revised CRISS, including (a) no major SSc-related events as defined by the revised CRISS; (b) a ≥5% improvement in predicted %FVC compared to baseline; and (c) a ≥25% reduction in mRSS, HAQ-DI, PtGA, and PGA compared to baseline. The objectives are to evaluate the proportion of subjects to achieve the objective; (4) changes in lung function as measured by predicted FVC% and corrected DLCO% at 12, 24, 52, 80, 104, 130, and 156 weeks after CAB-001 infusion; (5) changes in quantification by HRCT at 12, 24, 52, and 104 weeks after CAB-001 infusion; (6) changes in mRSS score; (7) changes in serum CRP and ESR; (8) the proportion of subjects who develop new terminal organ failure or whose terminal organ failure worsens, including lung disease, heart disease, and kidney disease; and (9) the effect of CAB-001 on disease activity of SSc as measured by changes in SSc-related antibodies.

[0345] The additional objective of this trial is to evaluate the effect of CAB-001 on immunosuppressive therapy and other combination SSc-related therapies, as measured by the proportion of subjects who do not require immunosuppressive therapy after CAB-001 infusion.

[0346] The additional purpose of this trial is to evaluate the effect of CAB-001 on overall survival and event-free survival (defined as survival without major organ damage), as measured by overall survival and event-free survival.

[0347] An additional objective of this study is to evaluate the effects of CAB-001 on patient-reported outcomes, as measured using SHAQ, SSPRO, UCLA SCTC GIT, PtGA, FACIT-F, pain NRS, and / or QOL (SF-36 and EQ-5D-5L).

[0348] Test design The design of this study is summarized in Figure 4. This study includes the following two cohorts based on the most prominent features(s) of the SSc phenotype.

[0349] Severe skin cohort: Subjects meet only the criteria for skin lesions and do not meet the criteria for lung, cardiac, or renal lesions as defined in inclusion criteria (6).

[0350] Organ cohort: Subjects meet the criteria for lung, cardiac, or renal lesions as defined in inclusion criteria (6), regardless of skin lesions.

[0351] Each participant undergoes leukocyte apheresis, and a CAB-001 cell preparation is manufactured for each participant. In summary, autologous T cells are genetically modified ex vivo by transduction using a lentiviral vector. After the manufacturing process is complete, the drug product is formulated and cryopreserved.

[0352] Following leukocyte apheresis and cell production, participants will be administered a preconditioning regimen. The preconditioning regimen will be a standard preconditioning regimen (e.g., 25 mg / m² on days -5, -4, and -3). 2 Fludarabine is administered intravenously, and 1,000 mg / m² is given on day 3. 2 This may be cyclophosphamide administered intravenously, or it may be a preconditioning regimen consisting solely of cyclophosphamide (as described in Table 1, for example). CAB-001 is administered to participants by intravenous infusion in one of the following four doses: 1 × 10⁻¹⁶ 6 Individual cells / kg, 3×10 5 Individual cells / kg, 3×10 6Individual cells / kg, 1×10 7 Individual cells / kg.

[0353] Participants will be evaluated for CAB-001-related toxicity and its effects on their underlying conditions during the initial 28-day evaluation period. Participants will be monitored on the day of infusion, with subsequent visits approximately 5, 8, 15, 22, and 29 days after infusion. In addition to routine chemical and hematological tests, clinical laboratory tests for PK sampling, coagulation panels, CRS lab tests, serum cytokine assessments, and CAR T evaluations will be performed up to day 29. A skin punch biopsy will also be taken at the visit on day 22. After the evaluation on day 29 after infusion, participants will be evaluated approximately once every 4 weeks (±7 days) in the first year, approximately every 12 weeks (±14 days) in the second year, and approximately every 26 weeks (±30 days) in the third year. All participants will be evaluated for safety, tolerability, efficacy, and quality of life for 156 weeks after infusion. Participants may also be monitored as needed for 15 years after treatment.

[0354] Inclusion and exclusion criteria The inclusion criteria for participation in this trial include: (1) being able to provide informed consent; (2) being between 18 and 70 years of age; (3) having a diagnosis of focal or diffuse SSc as defined by the 2013 American College of Rheumatology and European League Against Rheumatism classification criteria; (4) having had the first non-Raynaud's phenomenon symptom within the past seven years; (5) being inadequately treated with or intolerant to at least two immunomodulatory drugs for previous SSc treatments (in order for a subject to be eligible). (6) Documented evidence of a significant lesion of the skin, lungs, kidneys, or heart, defined as meeting one or more of the following conditions: I. Severe skin cohort subjects are subject to treatment with at least two of the following drugs for at least 12 weeks: azathioprine, corticosteroids, CY, hydroxychloroquine, IVIg, Janus kinase (JAK) inhibitors, mycophenolate mofetil or mycophenolate, methotrexate, rituximab, and / or tocilizumab); (6) Documented evidence of a significant lesion of the skin, lungs, kidneys, or heart, defined as meeting one or more of the following conditions: I. Severe skin cohort subjects are subject to treatment with at least two of the following drugs for at least 12 weeks: azathioprine, corticosteroids, CY, hydroxychloroquine, IVIg, Janus kinase (JAK) inhibitors, mycophenolate mofetil or mycophenolate, methotrexate, rituximab, and / or tocilizumab; (7) Documented evidence of a significant lesion of the skin, lungs, kidneys, or heart, defined as meeting one or more of the following conditions: I. Severe skin cohort subjects are subject to treatment with at least two of the following drugs for at least 12 weeks: azathioprine, corticosteroids, CY, hydroxychloroquine, IVIg, Janus kinase (JAK) inhibitors, mycophenolate mofetil or mycophenolate, methotrexate, rituximab, and / or tocilizumab; (8) Documented evidence of a significant lesion of the skin, lungs, kidneys, or heart, defined as meeting one or more of the following conditions: I. Severe skin cohort subjects are subject to treatment with at least two of the following drugs for at least 12 weeks; Only the modified criteria must be met, and the skin lesion criteria are all of the following: (a) Modified Rodnan Skin Score (mRSS) score > 15 and < 40, Health Assessment Questionnaire Disability Index (HAQ-DI) > 1.0, or elevated erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP), and (c) duration of illness less than 3 years and rapid progression defined as a 5-unit increase in mRSS over 12 months; II. Organ cohort subjects are (IIa) renal lesions, (IIb) cardiac lesions, or (IIc) Must meet one of the criteria for lung lesions, and may or may not meet the criteria for skin lesions: IIa. Renal lesion criteria: History of a non-active renal crisis at the time of screening, and stable serum creatinine (no increase of 20% or more) for at least 24 weeks after the renal crisis at the time of screening; IIb. Cardiac lesion criteria: Clinically stable condition with or without medication, and reasonable exclusion of non-scleroderma-related cardiac dysfunction (meeting one or more of the following: (a) well-controlled New York Heart Association (NYHA) class I or II heart disease; (b) atrial or ventricular arrhythmia; (c) second or third degree atrioventricular block; and (d) myocarditis); IIIc.Lung lesion criteria: The subject must have either (i) or (ii): (i) Forced vital capacity (FVC) > 55% and < 80% of predicted value, or hemoglobin-corrected pulmonary carbon monoxide diffusion capacity (DLCO) > 45% and < 80% of predicted value, and ILD on high-resolution computed tomography of the chest, or (ii) Rapidly progressive fibrotic ILD that has developed within the past year and is unexplained, as defined by two of the following three criteria: (Sub-criterion 1) Worsening of respiratory symptoms, (Sub-criterion 2) F within the past year of observation. Physiological evidence of disease progression, either by an absolute decrease of 5% or more in the predicted VC value, or an absolute decrease of 10% or more in the predicted hemoglobin-corrected DLCO value within one year of follow-up, and (Sub-criterion 3) radiological evidence of disease progression, indicated by one or more of the following: i. increased extent or severity of traction bronchiectasis and bronchiolectasis, ii. new ground-glass opacities with traction bronchiectasis, iii. new fine reticular opacities, iv. increased extent or coarser reticular opacities, v. new or enlarged honeycomb opacities, vi.(7) Increased reduction in lung lobe volume; (8) In the judgment of the investigator, the subject is a suitable candidate for the study, taking into account the potential benefits of other available treatments; (9) For immunocompromised individuals, all recommended vaccinations have been received prior to or during screening in accordance with the guidelines of the U.S. Centers for Disease Control and Prevention (CDC) or the facility (live vaccines must be administered at least 30 days prior to the pre-infusion visit, and non-live vaccines must be administered to the subject at least 2 weeks prior to the start of the investigational drug infusion, and, if possible, non-live vaccines must be administered at least 2 weeks prior to leukocyte apheresis); (10) Clinical stability as determined by the investigator; (11) If the subject is currently receiving standard immunosuppressive therapy, that therapy must be (11) The dose must be started at least 12 weeks prior to screening and remain stable for at least 8 weeks prior to the screening visit, except for dose reductions due to safety or tolerability reasons; (12) Fertile women must agree to use two acceptable methods of contraception from screening until at least 52 weeks after CAB-001 injection; (13) Both fertile, sexually active men and vasectomized, sexually active men receiving CAB-001 must use spermicidal condoms from screening until at least 52 weeks after CAB-001 injection, and fertile men must also agree not to provide sperm until at least 52 weeks after screening until at least 52 weeks after CAB-001 injection.

[0355] Exclusion criteria include patients with any of the following criteria: (1) a primary diagnosis of rheumatic AD other than SSc as determined by the investigator, in accordance with the investigator's judgment, including, but not limited to, systemic sclerosis-like disease, rheumatoid arthritis, SLE, polymyositis, dermatomyositis, systemic vasculitis, Sjögren's syndrome, antisynthesis syndrome, or mixed connective tissue disease (secondary Sjögren's syndrome or scleroderma myopathy are acceptable); (2) 2. Severe pulmonary dysfunction as defined by any of a-c below: a. Resting oxygen saturation without oxygen supplementation <92%, b. Predicted FVC ≤55%, c. Predicted DLCO after hemoglobin correction ≤45%; (3) Severe PAH as defined by mean pulmonary artery pressure at rest ≥25 mmHg on right heart catheterization (RHC) (subjects who have undergone RHC showing severe PAH within 12 months of screening are ineligible, and all other subjects will undergo screening echocardiography and if PAH is suggested) RHC is required if any of the following echocardiographic findings are observed: i. peak tricuspid regurgitation velocity > 2.8 m / sec, ii. elevated right ventricular systolic pressure, iii. abnormalities in right atrial size, shape, or wall thickness, iv. abnormalities in right ventricular size, shape, or wall thickness, and v. abnormalities in septal shape); (4) serious cardiac disease including any of the following: a. unstable angina or myocardial infarction or coronary artery bypass grafting within 24 weeks prior to leukocytosis, b. uncontrolled angina (5) A clinically significant arrhythmia, c. Clinical evidence of NYHA class III or IV CHF, d. Moderate to severe left ventricular dysfunction (left ventricular ejection fraction <45% on screening echocardiography), or e. A history of pacemaker or defibrillator implantation; (5) A serious renal disease including any of the following: a. Estimated glomerular filtration rate <60 mL / min / 1.73m2 using the formula of the Chronic Kidney Disease Epidemiological Collaborative Study, b. Scleroderma renal crisis within the past 24 weeks, or c.(6) Clinical evidence of active aspiration as determined by the investigator; (7) Alanine aminotransferase, aspartate aminotransferase, or bilirubin levels greater than twice the upper limit of normal; (8) A documented history of seizures, encephalopathy, or cerebral edema; (9) Active antral telangiectasia (GAVE, "watermelon stomach") on esophagogastroduodenoscopy (EGD) at screening (subjects with a negative EGD record within 24 weeks of screening, or those whose GAVE treatment was successfully confirmed by EGD within 24 weeks of treatment are eligible); (10) Hematological abnormalities as defined by a-d: a. Absolute lymphocyte count <500 cells / μL, b. Absolute neutrophil count <1,000 cells / μL, c. Platelet count <80,000 cells / μL d. Hemoglobin <8.0 g / dL; (11) Subjects who have received any of the following a-i: a. Prednisone at doses greater than 10 mg / day within 2 weeks prior to screening, b. More than two complications in the past 24 weeks, e.g., prednisone or equivalent at doses of 1 mg / kg / day for more than 5 days for asthma, or c. Rituximab or other B-cell depletion agents within 24 weeks prior to screening, or within 12 weeks if there is a test result indicating the presence of CD19+ B cells, d. Abatacept within 8 weeks prior to screening, e. IL-6 inhibitor (e.g., tocilizumab) within 12 weeks prior to screening, f. TNF inhibitor within 12 weeks prior to screening, g. Leflunomide within 8 weeks prior to screening, h. Therapeutic ferresis within 4 weeks prior to screening, i.(12) Any investigational drug with a half-life of 4 weeks or less than 5 weeks (whichever is longer); (13) A known malignancy or history of malignancy within the past 5 years, excluding excised / cured focal basal cell carcinoma or focal squamous cell carcinoma or cervical intraepithelial neoplasia; (14) A major surgery scheduled during the study evaluation period; (15) Contraindications to leukocytosis; (16) A history of anaphylactic or severe systemic reaction to FLU, CY, or any of their metabolites; (17) A positive test for human immunodeficiency virus, hepatitis C antibody, or hepatitis B surface antigen at screening, or evidence of active or chronic tuberculosis; (18) An active infection requiring medical intervention at screening; (19) Any inflammatory autoimmune disorder other than SSc requiring immunosuppressive therapy; (10) Severe, progressive, or uncontrolled kidney, liver, hematological, or gastrointestinal disease, including severe and uncontrolled infections such as sepsis and opportunistic infections. (20) If you currently have symptoms of lung disease, mental illness, heart disease, neurological disease, or brain disease; (21) If you have a history of CAR T-cell therapy; (22) If you have a history of solid organ (heart, liver, kidney, lung) transplant or hematopoietic stem cell transplant; (23) If you are unable or unwilling to comply with the protocol.

[0356] The combination of preconditioning regimens and CD19-CAR T-cell therapy is expected to be effective in treating systemic sclerosis.

[0357] Example 5 This example describes a Phase 1 / 2 open-label trial designed to evaluate the safety and efficacy of CAB-001 in participants with active idiopathic inflammatory muscle disease (IIM or myositis).

[0358] background Myositis (IIM) is a heterogeneous group of rare, acquired systemic autoimmune diseases characterized by inflammation and muscle weakness. Symptoms of IIM are diverse and may include muscle weakness, fatigue, dysphagia, and dyspnea. In some cases, IIM can affect other organs and systems of the body, such as the lungs, heart, or skin. Due to pain and muscle weakness, IIM patients may experience increased difficulty in activities requiring the use of proximal muscles, such as standing up from a chair, climbing stairs, or lifting objects (Malik et al. (2016) Front.Neurol.7:64). Extramuscular disease activity may include fever, arthralgia, arrhythmias, or cardiac dysfunction, and pulmonary complications often resulting from interstitial lung disease (ILD) (Lundberg et al. (2021) Nat.Rev.Dis.Primers 7(1):86).

[0359] Several subtypes of IIM that share inflammation and proximal muscle weakness include dermatomyositis (DM), antisynthesis syndrome (ASyS), and immune-mediated necrotizing myopathy (IMNM). These IIM subtypes typically affect middle-aged individuals, particularly women. DM, the most common subtype, has a prevalence of approximately 34,000 people in the United States (Kronzer et al. (2023) Arthritis Care Res. (Hoboken) 75(2):348-355) and is characterized by a distinctive rash in addition to muscle weakness. Antisynthesis syndrome affects approximately 10,000 people in the United States and is characterized by autoantibodies against aminoacyltransferase (tRNA) synthase, which cause inflammation and muscle weakness in addition to systemic symptoms such as intravascular disease (ILD), which is observed in 65-100% of patients (Iaccarino et al. (2014) J.Autoimmun. 48-49:122-127). IMNM, which affects approximately 6,000 people in the United States, is characterized by muscle tissue death and weakness (Allenbach et al. (2020) Nat. Rev. Rheumatol. 16(12):689-701). All three subtypes can result in...

Claims

1. A method for treating autoimmune diseases in subjects who require treatment for autoimmune diseases, (a) 100-1,500mg / m ２ Administering a preconditioning regimen consisting of one or more doses of cyclophosphamide per day to the subject, (b) Administering a therapeutically effective amount of genetically modified CAR immune cells to the subject The method comprising the subject receiving the cyclophosphamide at least three days prior to step (b).

2. The aforementioned preconditioning regimen is 1,000 mg / m² ２ The method according to claim 1, comprising administering a total dose of less than 100% of cyclophosphamide to the subject.

3. The aforementioned single dose of cyclophosphamide is 100 to 800 mg / m². ２ The method according to claim 1 or claim 2, wherein the value is per day.

4. A method for treating autoimmune diseases in subjects who require treatment for autoimmune diseases, (a) 100-800mg / m ２ Administering a preconditioning regimen to the subject containing one or more doses of cyclophosphamide per day, (b) Administering a therapeutically effective amount of genetically modified CAR immune cells to the subject The preconditioning regimen contains 1,000 mg / m² ２ The method comprising administering a total dose of less than a certain amount of cyclophosphamide to the subject.

5. The method according to claim 4, wherein the subject receives the cyclophosphamide at least three days before step (b).

6. A method for treating autoimmune diseases in subjects who require treatment for autoimmune diseases, (a) 100-1,500mg / m ２ Administering a preconditioning regimen to the subject containing one or more doses of cyclophosphamide per day, (b) Administering a therapeutically effective amount of genetically modified CAR immune cells to the subject comprising, wherein the genetically engineered CAR immune cells comprise a nucleic acid encoding a CAR, and the CAR comprises complementarity determining regions CDR Ｈ１ , CDR Ｈ２ , and CDR Ｈ３ comprising a heavy chain variable domain (V Ｈ ), and complementarity determining regions CDR Ｌ１ , CDR Ｌ２ , and CDR Ｌ３ comprising a light chain variable domain (V Ｌ ), and an extracellular antigen-binding site, wherein the CDR Ｈ１ , CDR Ｈ２ , and CDR Ｈ３ each comprise the amino acid sequences of SEQ ID NO: 1, 2, and 3, and the CDR Ｌ１ , CDR Ｌ２ , and CDR Ｌ３ each comprise the amino acid sequences of SEQ ID NO: 5, 6, and 7, said method.

7. The method according to claim 6, wherein the subject receives the cyclophosphamide at least three days before step (b).

8. The aforementioned preconditioning regimen is 1,000 mg / m² ２ The method according to claim 6 or 7, comprising administering a total dose of less than 100% of cyclophosphamide to the subject.

9. The aforementioned dose of cyclophosphamide is 100 to 800 mg / m². ２ The method according to any one of claims 6 to 8, wherein the value is per day.

10. The aforementioned single dose of cyclophosphamide is 1,000 mg / m². ２ The method according to any one of claims 1, 6, or 7, wherein the amount is per day.

11. The aforementioned single dose of cyclophosphamide is 750 mg / m². ２ The method according to any one of claims 1 to 9, wherein the value is per day.

12. The aforementioned single dose of cyclophosphamide is 500 mg / m². ２ The method according to any one of claims 1 to 9, wherein the value is per day.

13. The aforementioned single dose of cyclophosphamide is 375 mg / m². ２ The method according to any one of claims 1 to 9, wherein the value is per day.

14. The aforementioned single dose of cyclophosphamide is 250 mg / m². ２ The method according to any one of claims 1 to 9, wherein the value is per day.

15. The method according to any one of claims 1 to 14, wherein the subject is administered cyclophosphamide 3 to 7 days prior to step (b).

16. The method according to any one of claims 1 to 15, wherein the subject is administered cyclophosphamide three days prior to step (b).

17. The method according to any one of claims 1 to 9, wherein the preconditioning regimen includes administering cyclophosphamide to the subject three and four days prior to step (b).

18. The method according to any one of claims 1 to 9 or 17, wherein the preconditioning regimen essentially comprises administering cyclophosphamide to the subject three and four days prior to step (b).

19. The aforementioned single dose of cyclophosphamide is 250 mg / m². ２ / day, 375mg / m ２ / day, or 500 mg / m² ２ The method according to claim 17 or claim 18, wherein the amount is per day.

20. The method according to any one of claims 1 to 9 or 17, wherein the preconditioning regimen includes administering cyclophosphamide to the subject 3, 4, and 5 days prior to step (b).

21. The method according to any one of claims 1 to 9, 17, or 20, wherein the preconditioning regimen essentially comprises administering cyclophosphamide to the subject 3, 4, and 5 days prior to step (b).

22. The aforementioned dose of cyclophosphamide is 250 mg / m². ２ The method according to claim 20 or claim 21, wherein the value is per day.

23. The method according to any one of claims 1 to 22, wherein the subject is not administered fludarabine during the five days prior to step (b).

24. The method according to any one of claims 1 to 23, wherein the subject is not administered fludarabine during the seven days prior to step (b).

25. The method according to any one of claims 4 to 9, wherein fludarabine is not administered as part of the preconditioning regimen.

26. The method according to any one of claims 1 to 25, wherein the subject is not administered fludarabine.

27. The method according to any one of claims 1, 4, or 7, wherein the preconditioning regimen is one of the regimens disclosed in Table 1.

28. The aforementioned therapeutically effective amount of genetically modified CAR immune cells is 1 × 10 ５ Individual cells / kg ~ 1×10 ８ The method according to any one of claims 1 to 27, wherein the amount is individual cells / kg.

29. The aforementioned therapeutically effective amount of genetically modified CAR immune cells is 1 × 10 ６ Individual cells / kg ~ 1×10 ７ The method according to any one of claims 1 to 28, wherein the amount is individual cells / kg.

30. The method according to any one of claims 1 to 5, wherein the genetically modified CAR immune cells express CARs that specifically bind to B cell surface antigens.

31. The method according to claim 30, wherein the B cell surface antigen is CD19.

32. The genetically modified CAR immune cells contain nucleic acids that encode CAR, and the CAR is (i) an extracellular domain containing an antigen-binding site, (ii) Transmembrane domain and (iii) Co-stimulation domain and (iv) Intracellular signaling domain and The method according to any one of claims 1 to 5 or 30 to 31, including the method described in claim 1 to 5 or 30 to 31.

33. The antigen-binding site is the complementarity-determining region CDR. Ｈ１ , CDR Ｈ２ , and CDR Ｈ３ Heavy chain variable domain (V Ｈ ) and the complementarity determination region CDR Ｌ１ , CDR Ｌ２ , and CDR Ｌ３ Light chain variable domain (V Ｌ ) and the CDR Ｈ１ , CDR Ｈ２ , and CDR Ｈ３ However, each contains the amino acid sequences of Sequence ID No. 1, 2, and 3, respectively, and the CDR Ｌ１ , CDR Ｌ２ , and CDR Ｌ３ The method according to claim 32, wherein the amino acid sequences are those of sequence numbers 5, 6, and 7, respectively.

34. The method according to any one of claims 6 to 9, wherein the CAR further comprises a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

35. The method according to any one of claims 6 to 9 or 32 to 34, wherein the antigen-binding site is a humanized antigen-binding site or a fully human antigen-binding site.

36. The aforementioned V Ｈ The method according to any one of claims 6 to 9 or 33 to 35, wherein VL contains an amino acid sequence that is at least 95% identical to the amino acid sequences of SEQ ID NOs. 4 and 8, respectively.

37. The aforementioned V Ｈ and V Ｌ The method according to any one of claims 6 to 9 or 33 to 36, wherein the amino acid sequences are those of SEQ ID NOs: 4 and 8, respectively.

38. The method according to any one of claims 6 to 9 or 32 to 37, wherein the antigen-binding site is located in scFv.

39. The method according to claim 38, wherein the scFv contains the amino acid sequence of SEQ ID NO:

9.

40. The method according to any one of claims 32 to 39, wherein the transmembrane domain includes a CD8α chain transmembrane domain.

41. The method according to claim 40, wherein the CD8α-chain transmembrane domain comprises the amino acid sequence of SEQ ID NO:

19.

42. The method according to any one of claims 32 to 41, wherein the costimulatory domain includes a 4-1BB intracellular domain.

43. The method according to claim 42, wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO:

20.

44. The method according to any one of claims 32 to 43, wherein the intracellular signaling domain includes a CD3ζ signaling domain.

45. The method according to claim 44, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:

21.

46. The method according to any one of claims 32 to 45, wherein the CAR further comprises a hinge domain or linker positioned between the antigen-binding site and the transmembrane domain.

47. The method according to claim 46, wherein the hinge domain is a CD8α chain hinge.

48. The method according to claim 47, wherein the CD8α chain hinge comprises the amino acid sequence of SEQ ID NO:

22.

49. The method according to any one of claims 6 to 9 or 32 to 48, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

23.

50. The method according to any one of claims 1 to 49, wherein the autoimmune disease is a B-cell mediated autoimmune disease.

51. The method according to any one of claims 1 to 50, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myasthenia gravis (MG), myositis, and membranous nephropathy.

52. The method according to any one of claims 1 to 51, wherein the autoimmune disease is selected from the group consisting of lupus nephritis, SLE with anti-dsDNA antibodies, mucosal PV, mucocutaneous PV, MuSK-related MG, AChR MG, anti-synthesis syndrome, dermatomyositis, and immune-mediated necrotizing myopathy.

53. The method according to any one of claims 1 to 52, wherein the autoimmune disease is SLE.

54. The method according to any one of claims 1 to 53, wherein the CAR immune cells are CAR T cells or CAR NK cells.

55. The method according to any one of claims 1 to 54, wherein the CAR immune cells are CAR T cells.

56. A method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease, the method comprising administering genetically modified immune cells containing nucleic acids encoding a CAR, wherein the CAR is (i) an extracellular domain containing an antigen-binding site that binds to CD19, (ii) Transmembrane domain and (iii) Co-stimulation domain and (iv) Intracellular signaling domain and The antigen-binding site is the complementarity-determining region CDR. Ｈ１ , CDR Ｈ２ , and CDR Ｈ３ Heavy chain variable domain (V Ｈ ) and the complementarity determination region CDR Ｌ１ , CDR Ｌ２ , and CDR Ｌ３ Light chain variable domain (V Ｌ ) and the CDR Ｈ１ , CDR Ｈ２ , and CDR Ｈ３ However, each contains the amino acid sequences of Sequence ID No. 1, 2, and 3, respectively, and the CDR Ｌ１ , CDR Ｌ２ , and CDR Ｌ３ The method, wherein each amino acid sequence is the sequence of sequence numbers 5, 6, and 7, respectively.

57. The method according to claim 56, wherein the antigen-binding site is a humanized antigen-binding site or a fully human antigen-binding site.

58. The aforementioned V Ｈ The method according to claim 56 or claim 57, wherein VL contains an amino acid sequence that is at least 95% identical to the amino acid sequences of SEQ ID NOs. 4 and 8, respectively.

59. The aforementioned V Ｈ and V Ｌ The method according to any one of claims 56 to 58, wherein the amino acid sequences are those of sequence numbers 4 and 8, respectively.

60. The method according to any one of claims 56 to 59, wherein the antigen-binding site is located in scFv.

61. The method according to claim 60, wherein the scFv contains the amino acid sequence of SEQ ID NO:

9.

62. The method according to any one of claims 56 to 61, wherein the transmembrane domain includes a CD8α chain transmembrane domain.

63. The method according to claim 62, wherein the CD8α-chain transmembrane domain comprises the amino acid sequence of SEQ ID NO:

19.

64. The method according to any one of claims 56 to 63, wherein the costimulatory domain includes a 4-1BB intracellular domain.

65. The method according to claim 64, wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO:

20.

66. The method according to any one of claims 56 to 65, wherein the intracellular signaling domain includes a CD3ζ signaling domain.

67. The method according to claim 66, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:

21.

68. The method according to any one of claims 56 to 67, wherein the CAR further comprises a hinge or linker positioned between the antigen-binding site and the transmembrane domain.

69. The method according to claim 68, wherein the hinge domain is a CD8α chain hinge.

70. The method according to claim 69, wherein the CD8α chain hinge comprises the amino acid sequence of SEQ ID NO:

22.

71. The method according to any one of claims 56 to 70, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

23.

72. The method according to any one of claims 56 to 71, wherein the immune cells are T cells or NK cells.

73. The method according to any one of claims 56 to 72, wherein the immune cell is a T cell.

74. The method according to claim 73, wherein the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, αβ T cells, and γδ T cells.

75. The method according to any one of claims 56 to 74, wherein the immune cells are administered to the subject with a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient.

76. The aforementioned genetically modified immune cells 1 × 10 ５ Individual cells / kg ~ 1×10 ８ The method according to any one of claims 56 to 75, comprising administering to the subject at a dose of individual cells / kg.

77. The above dose is 1 × 10 ６ Individual cells / kg ~ 1×10 ７ The method according to claim 76, wherein the amount is individual cells / kg.

78. The method according to any one of claims 56 to 77, wherein the autoimmune disease is a B-cell mediated autoimmune disease.

79. The method according to any one of claims 56 to 78, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris (PV), myasthenia gravis (MG), myositis, and membranous nephropathy.

80. The method according to any one of claims 56 to 79, wherein the autoimmune disease is selected from the group consisting of lupus nephritis, SLE with anti-dsDNA antibodies, mucosal PV, mucocutaneous PV, MuSK-related MG, AChR MG, anti-synthesis syndrome, dermatomyositis, and immune-mediated necrotizing myopathy.

81. The method according to any one of claims 56 to 80, wherein the autoimmune disease is SLE.

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